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Molecular Biology Fifth Edition Chapter 1 A Brief History Lecture PowerPoint to accompany Robert F. Weaver Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 1-2 A Brief History • What is molecular biology? – The attempt to understand biological phenomena in molecular terms – The study of gene structure and function at the molecular level • Molecular biology is a melding of aspects of genetics and biochemistry 1-3 1.1 Transmission Genetics • Transmission genetics deals with the transmission of traits from parental organisms to their offspring • The chemical composition of genes was not known until 1944 – Gene - genetic units – Phenotype - observable characteristics 1-4 Mendel’s Laws of Inheritance • A gene can exist in different forms called alleles • One allele can be dominant over the other, recessive, allele • The first filial generation (F1) contains offspring of the original parents • If each parent carries two copies of a gene, the parents are diploid for that gene 1-5 Mendel’s Laws of Inheritance • Homoozygotes have two copies of the same allele • Heterozygotes have one copy of each allele • Parents in 1st mating are homozygotes, having 2 copies of one allele • Sex cells, or gametes, are haploid, containing only 1 copy of each gene • Heterozygotes produce gametes having either allele • Homozygotes produce gametes having only one allele 1-6 Summary • Genes can exist in several different forms or alleles • One allele can be dominant over the other, so heterozygotes having two different alleles of one gene will generally exhibit the characteristic dictated by the dominant allele • The recessive allele is not lost; it can still exert its influence when paired with another recessive allele in a homozygote 1-7 The Chromosome Theory of Inheritance • Chromosomes are discrete physical entities that carry genes • Thomas Hunt Morgan used the fruit fly, Drosophila melanogaster, to study genetics • Autosomes occur in pairs in a given individual (not the X or the Y chromosome) • Sex chromosomes are identified as X and Y – Females have two X chromosomes – Males have one X and one Y chromosome 1-8 Location of Genes on a Chromosome • Every gene has its place, or locus, on a chromosome • Genotype is the combination of alleles found in an organism • Phenotype is the visible expression of the genotype – Wild-type phenotype is the most common or generally accepted standard – Mutant alleles are usually recessive 1-9 Genetic Recombination and Mapping • In early experiments genes on separate chromosomes behaved independently • Genes on the same chromosome behaved as if they were linked • This genetic linkage is not absolute • Offspring show new combinations of alleles not seen in the parents when recombination occurs 1-10 Recombination • During meiosi ...

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Molecular Biology Fifth Edition Chapter 1 A Brief History Lecture PowerPoint to accompany Robert F. Weaver Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 1-2 A Brief History • What is molecular biology? – The attempt to understand biological phenomena in molecular terms – The study of gene structure and function at the molecular level • Molecular biology is a melding of aspects of genetics and biochemistry
1-3 1.1 Transmission Genetics • Transmission genetics deals with the transmission of traits from parental organisms to their offspring • The chemical composition of genes was not known until 1944 – Gene - genetic units – Phenotype - observable characteristics 1-4 Mendel’s Laws of Inheritance • A gene can exist in different forms called alleles • One allele can be dominant over the other, recessive, allele • The first filial generation (F1) contains offspring of the original parents • If each parent carries two copies of a gene, the parents are diploid for that gene
1-5 Mendel’s Laws of Inheritance • Homoozygotes have two copies of the same allele • Heterozygotes have one copy of each allele • Parents in 1st mating are homozygotes, having 2 copies of one allele • Sex cells, or gametes, are haploid, containing only 1 copy of each gene • Heterozygotes produce gametes having either allele • Homozygotes produce gametes having only one allele 1-6 Summary • Genes can exist in several different forms or alleles • One allele can be dominant over the other, so heterozygotes having two different alleles of one gene will generally exhibit the characteristic dictated by the dominant allele • The recessive allele is not lost; it can still exert its influence when paired with another recessive allele
in a homozygote 1-7 The Chromosome Theory of Inheritance • Chromosomes are discrete physical entities that carry genes • Thomas Hunt Morgan used the fruit fly, Drosophila melanogaster, to study genetics • Autosomes occur in pairs in a given individual (not the X or the Y chromosome) • Sex chromosomes are identified as X and Y – Females have two X chromosomes – Males have one X and one Y chromosome 1-8 Location of Genes on a Chromosome • Every gene has its place, or locus, on a chromosome • Genotype is the combination of alleles found in an organism • Phenotype is the visible expression of the genotype – Wild-type phenotype is the most common or
generally accepted standard – Mutant alleles are usually recessive 1-9 Genetic Recombination and Mapping • In early experiments genes on separate chromosomes behaved independently • Genes on the same chromosome behaved as if they were linked • This genetic linkage is not absolute • Offspring show new combinations of alleles not seen in the parents when recombination occurs 1-10 Recombination • During meiosis, gamete formation, crossing over can occur resulting in the exchange of genes between the two homologous chromosomes • The result of the crossing-over event produces a new combination of alleles
• This process is called recombination 1-11 Genetic Mapping • Morgan proposed that the farther apart two genes are on a chromosome, the more likely they are to recombine • If two loci recombine with a frequency of 1%, they are said to be separated by a map distance of one centimorgan (named for Morgan) • This mapping observation applies both to prokaryotes and to eukaryotes 1-12 Physical Evidence for Recombination • Microscopic examination of the maize chromosome provided direct physical observation of recombination using easily identifiable features of one chromosome • Similar observations were made in Drosophila • Recombination was detected both
physically and genetically in both animals and plants 1-13 Summary • The chromosome theory of inheritance holds that genes are arranged in linear fashion on chromosomes • Certain traits tend to be inherited together when the genes for those traits are on the same chromosome • Recombination between two homologous chromosomes during meiosis can scramble the parental alleles to yield nonparental combinations • The farther apart two genes are on a chromosome the more likely it is that recombination will occur 1-14 1.2 Molecular Genetics • The Discovery of DNA: The general structure of nucleic acids was discovered by the end of the 19th century
– Long polymers or chains of nucleotides – Nucleotides are linked by sugars through phosphate groups • Composition of Genes: DNA? RNA? Protein? In 1944, Avery and his colleagues demonstrated that genes are composed of DNA 1-15 The Relationship between Genes and Proteins • Experiments have shown that a defective gene gives a defective or absent enzyme • This lead to the proposal that one gene is responsible for making one enzyme • Proposal not quite correct for 3 reasons: 1. One enzyme may be composed of several polypeptides, each gene codes for only one polypeptide 2. Many genes code for non-enzyme proteins 3. End products of some genes are not polypeptides (i.e. tRNA, rRNA)
1-16 Activities of Genes Genes perform three major roles • Replicated faithfully • Direct the production of RNAs and proteins • Accumulate mutations thereby allowing for evolution 1-17 Replication • Franklin and Wilkins produced x-ray diffraction data on DNA, Watson and Crick proposed that DNA is double helix – Two DNA strands wound around each other – Strands are complementary – if you know the sequence of one strand, you automatically know the sequence of the other strand • Semiconservative replication keeps one strand of the parental double helix conserved in each of the daughter double helices
1-18 Genes Direct the Production of Polypeptides • Gene expression is the process by which a gene product is made • Two steps are required – 1. Transcription: DNA is transcribed into RNA – 2. Translation: the mRNA is read or translated to assemble a protein – Codon: a sequence of 3 nucleic acid bases that code for one amino acid within the mRNA 1-19 Genes Accumulate Mutations Genes change in several ways • Change one base to another • Deletions of one base up to a large segment • Insertions of one base up to a large segment
• The more drastic the change, the more likely it is that the gene or genes involved will be totally inactivated 1-20 Summary • All cellular genes are made of DNA arranged in a double helix • This structure explains how genes replicate, carry information and collect mutations • The sequence of nucleotides in a gene is a genetic code that carries the information for making an RNA • A change in the sequence of bases constitutes and mutation, which can change the sequence of amino acids in the genes polypeptide product 1-21 1.3 The Three Domains of Life Current research theories support the division of living organisms into three domains 1. Bacteria
2. Eukaryota 3. Archaea • Like bacteria as they are organisms without nuclei • More similar to eukaryotes in the context of their molecular biology 1-22 Archaea Archaea live in the most inhospitable regions of the earth • Thermophiles tolerate extremely high temperatures • Halophiles tolerate very high salt concentrations • Methanogens produce methane as a by-product of metabolism Molecular Biology Fifth Edition Chapter 2 The Molecular Nature
of Genes Lecture PowerPoint to accompany Robert F. Weaver Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 2-2 The Nature of Genetic Material Historical Background • Miescher isolated nuclei from pus (white blood cells) in 1869 – Found a novel phosphorus-bearing substance = nuclein – Nuclein is mostly chromatin, a complex of DNA and chromosomal proteins • End of 19th century – DNA and RNA separated from proteins • Levene, Jacobs, et al. characterized basic composition of DNA and RNA 2-3
Molecular Foundation: Early experiments that explored the question: What is the genetic material? • Key experiments performed by Frederick Griffith in 1928 • Observed change in Streptococcus pneumoniae — from avirulent (R) rough colonies, bacteria without capsules, to virulent (S) smooth colonies, bacteria that had capsules • Result: Heat-killed virulent bacteria could transform avirulent bacteria into virulent bacteria 2-4 Outline of Griffith’s Transformation Experiments 2-5 DNA: The Transforming Material In 1944 Avery, Macleod and McCarty used a transformation test similar to Griffith’s procedure taking care to define the chemical
nature of the transforming substance – Techniques used excluded both protein and RNA as the chemical agent of transformation – Exclusion of DNA verified that DNA is the chemical agent of transformation of S. pneumoniae from avirulent to virulent 2-6 Analytical Tools Physical-chemical analysis has often used: 1. Ultracentrifugation Used to estimate size of material 2. Electrophoresis Indicated high charge-to-mass ratio 3. Ultraviolet Absorption Spectrophotometry Absorbance of UV light matched that of DNA 4. Elementary Chemical Analysis Nitrogen-to-phosphorus ratio of 1.67, expected for DNA but lower than expected for protein 2-7
Confirmation for DNA as the genetic material • In the 1940s geneticists doubted the use of DNA as the genetic material as it appeared to be monotonous repeats of 4 bases • By 1953 Watson & Crick published the double- helical model of DNA structure and Chargaff demonstrated that the 4 bases were not present in equal proportions • In 1952 Hershey and Chase demonstrated that bacteriophage infection comes from DNA, adding more evidence to support that DNA is the genetic material 2-8 Outline of Hershey and Chase’s Experiment 2-9 Summary • The classic molecular biology experiments performed by Griffith, Avery, MacLeod,
Mccarty, Hershey and Chase combined revealed that DNA is the genetic element 2-10 The Chemical Nature of Polynucleotides • Biochemists determined the components of nucleotides during the 1940s • The component parts of DNA – Nitrogenous bases: • Adenine (A) • Cytosine (C) • Guanine (G) • Thymine (T) – Phosphoric acid – Deoxyribose sugar 2-11 Nucleosides and Deoxyribose • RNA component parts – Nitrogenous bases • Like DNA except Uracil
(U) replaces Thymine – Phosphoric acid – Ribose sugar • Bases use ordinary numbers • Carbons in sugars are noted as primed numbers • Nucleotides contain phosphoric acid • Nucleosides lack the phosphoric acid • Deoxyribose lacks a hydroxyl group (OH) at the 2-position 2-12 Purines and Pyrimidines • Adenine and guanine are related structurally to the parent molecule purine • Cytosine, thymine and uracil resemble the parent molecule pyrimidine 2-13
DNA Linkage • Nucleotides are nucleosides with a phosphate group attached through a phosphodiester bond • Nucleotides may contain one, two, or even three phosphate groups linked in a chain 2-14 A Trinucleotide The example trinucleotide has polarity – The top of molecule has a free 5’-phosphate group = 5’ end – The bottom has a free 3’- hydroxyl group = 3’ end 2-15 Summary
• DNA and RNA are chain-like molecules composed of subunits called nucleotides • Nucleotides contain a base linked to the 1’-position of a sugar and a phosphate group • The phosphate joins the sugars in a DNA or RNA chain through their 5’- and 3’- hydroxyl groups by phosphodiester bonds 2-16 DNA Structure The Double Helix • Rosalind Franklin’s x-ray diffraction data suggested that DNA had a helical shape • The data also indicated a regular, repeating structure • Chargaff’s data revealed that the content of purines was always roughly equal to pyrimidines • Watson and Crick proposed a double helix with sugar-phosphate backbones on the outside and bases aligned on the interior 2-17
DNA Helix • Structure compared to a twisted ladder – Curving sides of the ladder represent the sugar- phosphate backbone – Ladder rungs are the base pairs – There are about 10 base pairs per turn • Arrows indicate that the two strands are antiparallel 2-18 Summary • The DNA molecule is a double helix, with sugar-phosphate backbones on the outside and base pairs on the inside • The bases pair in a specific way: – Adenine (A) with thymine (T) – Guanine (G) with cytosine (C) 2-19
Genes Made of RNA Viruses are a package of genes – No metabolic activity of their own – When a virus infects a host cell, the cellular machinery is diverted and begins to make viral proteins – Viral genes are replicated and used for the production of viral protein that assemble into virus particles Viruses contain nucleic acid, some viruses use DNA genes, but some viruses have RNA genes, either double- or single-stranded 2-20 Physical Chemistry of Nucleic Acids DNA and RNA molecules can appear in several different structural variants – Changes in relative humidity will cause variation in DNA molecular structure – The twist of the DNA molecule is normally shown to be right-handed, but left-handed DNA also exists and was identified in 1979
2-21 A Variety of DNA Structures • High humidity (92%) DNA is called the B-form • Reduce relative humidity to 75% and DNA takes on the A-form – Plane of base pairs in A- form is no longer perpendicular to the helical axis – The A-form is seen when one strand of DNA is hybridized with one strand of RNA strand • When wound in a left- handed helix, DNA is found in the Z-form • To date at least one gene requires Z-DNA for activation
2-22 Summary • In the cell, DNA may exist in the common B form, with horizontal base pairs • A very small fraction of the DNA may assume a left-handed helical form called the Z-form • An RNA-DNA hybrid assumes a third helical shape, called the A-form, with base pairs tilted away from the horizontal 2-23 Variation in DNA between Organisms • Ratios of G to C and A to T are fixed in any specific organism • The total percentage of G + C varies over a range of 22 to 73% • These reflect differences in physical
properties 2-24 DNA Denaturation or Melting • With heating, noncovalent forces holding DNA strands together weaken and break • When the forces break, the two strands come apart in denaturation or melting • The temperature at which the DNA strands are ½ denatured is the melting temperature or Tm • GC content of DNA has a significant effect on Tm with higher GC content yielding a higher Tm 2-25 DNA Denaturation • In addition to heat, DNA can be denatured by: – Organic solvents – High pH – Low salt concentration • GC content also affects DNA density
– Direct, linear relationship – Due to larger molar volume of A-T base pairs compared to G-C base pairs 2-26 Summary • The GC content of a natural DNA can vary from less than 25% to almost 75% • The GC content has a strong effect on the physical properties of the DNA, each of which increase linearly with GC content – The melting temperature, the temperature at which the two strands are half-dissociated or denatured – Density – Low ionic strength, high pH and organic solvents also promote DNA denaturation 2-27 DNA Renaturation
• After two DNA strands separate, under proper conditions the strands can come back together • Process is called annealing or renaturation • Three most important factors: – Temperature – best at about 25 C below Tm – DNA Concentration – within limits higher concentration better likelihood that 2 complementary will find each other – Renaturation Time – as increase time, more annealing will occur 2-28 Polynucleotide Chain Hybridization Hybridization is a process of putting together a combination of two different nucleic acids – Strands could be 1 DNA and 1 RNA – Also could be 2 DNA with complementary or nearly
complementary sequences 2-29 DNA Sizes DNA size is expressed in 3 different ways: – Number of base pairs – Molecular weight – 660 is molecular weight of 1 base pair – Length – 33.2 Å per helical turn of 10.4 base pairs DNA can be measured by electron microscopy or gel electrophoresis 2-30 DNAs of Various Sizes and Shapes • Bacterial DNA is typically circular • Some DNA will be linear • Supercoiled DNA coils or wraps around itself like a twisted rubber band
2-31 Summary • Natural DNAs come in sizes ranging from several kilobases to thousands of megabases • The size of a small DNA can be estimated by electron microscopy • This technique can also reveal whether a DNA is circular or linear and whether it is supercoiled 2-32 Relationship between DNA Size and Genetic Capacity How does one know how many genes are in a particular piece of DNA? – Can’t determine from DNA size alone – Factors include: • How much of the DNA is devoted to genes? • What is the space between genes? – One can estimate the upper limit of number genes a piece of DNA can hold
2-33 DNA Size and Genetic Capacity How many genes are in a piece of DNA? – Start with basic assumptions • Genes encode protein (ignoring the RNAs made) • The average protein is abut 40,000 D – How many amino acids does this represent? • Average mass of an amino acid is about 110 D • Average protein – 40,000 / 110 = 364 amino acids • Each amino acid = 3 DNA base pairs • 364 amino acids requires 1092 base pairs 2-34 DNA Genetic Capacity How large is an average piece of DNA? – E. coli chromosome • 4.6 x 106 bp • ~4200 proteins – Phage l (infects E. coli) • 4.85 x 104 bp • ~44 proteins – • 5375 bp • ~5 proteins (squeezes in more by overlapping
genes) 2-35 DNA Content and the C-Value Paradox • The C-value is the DNA content per haploid cell • One might expect that more complex organisms need more genes than simple organisms • For the mouse or human compared to yeast this is correct • Yet the frog has 7 times more genes per cell than humans 2-36 C-Value Paradox • The observation that more complex organisms will not always need more genes than simple organisms is called the C-value paradox • The most likely explanation for the paradox is that organisms with extraordinarily high C-values simply have
a great deal of extra, noncoding DNA 2-37 Summary • There is a rough correlation between DNA content and number of genes in a cell or virus • This correlation breaks down in several cases of closely related organisms where the DNA content per haploid cell (C-value) varies widely • The C-value paradox is probably explained not by extra genes, but by extra noncoding DNA in some organisms Molecular Biology Fifth Edition Chapter 3 An Introduction to Gene Function Lecture PowerPoint to accompany Robert F. Weaver
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 3-2 3.1 Storing Information Producing a protein from DNA involves both transcription and translation – A codon is the 3 base sequence that determines what amino acid is used – Template strand is the DNA strand that is used to generate the mRNA – Nontemplate strand is not used in transcription 3-3 Protein Structure Proteins are chain-like polymers of small subunits, called amino acids
– DNA has 4 different nucleotides (A,G, C, T) – Proteins have 20 different amino acids with: • An amino group • A hydroxyl group • A hydrogen atom • A specific side chain 3-4 Polypeptides • Amino acids are joined together via peptide bonds • Chains of amino acids are called polypeptides • Proteins are composed of 1 or more polypeptides • Polypeptides have polarity – Free amino group at one end is the amino- or N-terminus – Free hydroxyl group at the other end is the carboxyl- or C-terminus 3-5 Types of Protein Structure (4) • The linear order of amino acids is a protein’s primary structure • Interaction of the amino acids’ amino and carboxyl groups gives rise to the secondary structure of a protein – Secondary structure is the result of amino acid and
carboxyl group hydrogen bonding among near neighbors – Common types of secondary structure: -helix -sheet 3-6 Helical Secondary Structure • In a-helix secondary structure polypeptide backbone groups H bond with each other • The dashed lines indicate hydrogen bonds between nearby amino acids 3-7 Sheet Secondary Structure • The b-sheet pattern of 2° structure also occurs when
polypeptide backbone groups form H bonds • In the sheet configuration, extended polypeptide chains are packed side by side • This side-by-side packing creates a sheet appearance 3-8 Tertiary Structure • The total three- dimensional shape of a polypeptide is its tertiary structure • A prominent aspect of this structure is the interaction of the amino acid side chains • The globular form of a polypeptide is a roughly
spherical structure 3-9 Protein Domains • Compact structural regions of a protein are referred to as domains • Immunoglobulins provide an example of 4 globular domains • Domains may contain common structural-functional motifs – Zinc finger – Hydrophobic pocket • Quaternary structure is the interaction of 2 or more polypeptides 3-10 Summary • Proteins are polymers of amino acids linked through peptide bonds
• The sequence of amino acids in a polypeptide (primary structure) gives rise to that molecule’s: – Local shape (secondary structure) – Overall shape (tertiary structure) – Interaction with other polypeptides (quaternary structure) 3-11 Protein Function Proteins: – Provide the structure that help give cells integrity and shape – Serve as hormones carrying signals from one cell to another – Bind and carry substances – Control the activities of genes – Serve as enzymes that catalyze hundreds of chemical reactions 3-12 Relationship Between Genes and Proteins
• 1902 Dr. Garrod suggested a link between a human disease and a recessive gene • If a single gene controlled the production of an enzyme, lack of that enzyme could result in the buildup of homogentisic acid which is excreted in the urine • Should the gene responsible for the enzyme be defective, then the enzyme would likely also be defective 3-13 One-Gene/One-Polypeptide • Over time many experiments (i.e., Beadle and Tatum) have built on Garrod’s initial work • Many enzymes contain more than one polypeptide chain and each polypeptide is usually encoded in one gene • These observations have lead to the one gene one polypeptide hypothesis: Most genes contain the information for making one polypeptide 3-14 Information Carrier
• In the 1950s and 1960s, the concept that messenger RNA carries information from gene to ribosome was developed • An intermediate carrier was needed as DNA is found in the nucleus, while proteins are made in the cytoplasm • Therefore, some type of molecule must move the information from the DNA in the nucleus to the site of protein synthesis in the cytoplasm 3-15 Discovery of Messenger RNA • Ribosomes are the cytoplasmic site of protein synthesis • Jacob and colleagues proposed that messengers, an alternative of non- specialized ribosomes, translate unstable RNAs • These messengers are independent RNAs that move information from genes to ribosomes 3-16
Summary Messenger RNAs carry the genetic information from the genes to the ribosomes, which then synthesize polypeptides 3-17 Transcription • Transcription follows the same base- pairing rules as DNA replication – Remember U replaces T in RNA – This base-pairing pattern ensures that the RNA transcript is a faithful copy of the gene • For transcription to occur at a significant rate, its reaction is enzyme mediated • The enzyme directing transcription is called RNA polymerase 3-18 Synthesis of RNA 3-19
Phases of Transcription Transcription occurs in three phases: 1. Initiation 2. Elongation 3. Termination 3-20 Initiation • RNA polymerase recognizes a specific region, the promoter, which lies just upstream of gene • The polymerase binds tightly to the promoter causing localized separation of the two DNA strands • The polymerase starts building the RNA chain by adding ribonucleotides • After several ribonucleotides are joined together the enzyme leaves the promoter and elongation begins
3-21 Elongation • RNA polymerase directs the addition of ribonucleotides in the 5’ to 3’ direction • Movement of the polymerase along the DNA template causes the “bubble” of separated DNA strands to move also • As the RNA polymerase proceeds along the DNA, the two DNA strands that have opened for the “bubble” reform the double helix behind the transciptional machinery 3-22 Transcription and DNA Replication Two fundamental differences between transcription and DNA replication 1. RNA polymerase only makes one RNA strand during transcription, it copies only one DNA strand in a given gene – This makes transcription asymmetrical – Replication is semiconservative 2. DNA melting is limited and transient during
transcription, but the separation is permanent in replication 3-23 Termination • Analogous to the initiating activity of promoters, there are regions at the other end of genes that serve to terminate transcription • These terminators work with the RNA polymerase to loosen the association between the RNA product and the DNA template • As a result, the RNA dissociates from the RNA polymerase and the DNA and transcription stops 3-24 Important Note about Conventions • RNA sequences are written 5’ to 3’, left to right • Translation occurs 5’ to 3’ with ribosomes reading the message 5’ to 3’ • Genes are written so that transcription proceeds in
a left to right direction • The gene’s promoter area lies just before the start area, said to be upstream of transcription • Genes are therefore said to lie downstream of their promoters 3-25 Summary • Transcription takes place in three stages: – Initiation – Elongation – Termination • Initiation involves the binding of RNA polymerase to the promoter, local melting and forming the first few phosphodiester bonds • During elongation, the RNA polymerase links together ribonucleotides in the 5’ to 3’ direction to make the rest of the RNA • In termination, the polymerase and RNA product dissociate from the DNA template 3-26 Translation - Ribosomes
• Ribosomes are protein synthesizing machines – Ribosome subunits are designated with numbers such as 50S or 30S – Number is the sedimentation coefficient - a measure of speed with which the particles sediment through a solution spun in an ultracentrifuge based on mass and shape • Each ribosomal subunit contains RNA and protein 3-27 Ribosomal RNA • The two ribosomal subunits both contain ribosomal RNA (rRNA) molecules and a variety of proteins • rRNAs participate in protein synthesis but do NOT code for proteins • No translation of rRNA occurs 3-28 Summary
• Ribosomes are the cell’s protein factories • Bacteria contain 70S ribosomes • Each ribosome has 2 subunits – 50 S – 30 S • Each subunit contains rRNA and many proteins 3-29 tRNA: Translation Adapter Molecule • Generating protein from ribosomes requires change from the nucleic acid to amino acid • This change is described as translation from the nucleic acid base pair language to the amino acid language • Crick proposed that some type of adapter molecule was needed to provide the bridge for translation, perhaps a small RNA • The physical interface between the mRNA and the ribosome
3-30 Transfer RNA: Adapter Molecule • Transfer RNA is a small RNA that recognizes both RNA and amino acids • A cloverleaf model is used to illustrate tRNA structure • The 3’ end binds to a specific amino acid • The anticodon loop contains a 3 nucleotide sequence that pairs with complementarity to a codon in mRNA 3-31 Codons and Anticodons • Enzymes that catalyze attachment of amino acid to tRNA are aminoacyl- tRNA synthetases • A triplet in mRNA is called
a codon • The complementary sequence to a codon found in a tRNA is the anticodon 3-32 Summary • Two important sites on tRNAs allow them to recognize both amino acids and nucleic acids • One site binds covalently to an amino acid • The site contains an anticodon that base- pairs with a codon in the mRNA • tRNAs are capable of serving the adapter role and are the key to the mechanism of translation 3-33 Initiation of Protein Synthesis • The initiation codon (AUG) interacts with a special aminoacyl-tRNA
– In eukaryotes this is methionyl-tRNA – In bacteria it is a derivative called N-formylmethionyl- tRNA • Position of the AUG codon: – At start of message AUG is initiator – In middle of message AUG is regular methionine • Shine-Dalgarno sequence lies just upstream of the AUG, functions to attract ribosomes – Unique to bacteria – Eukaryotes have special cap on 5’-end of mRNA 3-34 Translation Elongation • During initiation the initiating aminoacyl-tRNA binds within the P site of the ribosome • Elongation adds amino acids one at a time to the initiating amino acid • The first elongation step is binding second aminoacyl-tRNA to the A site on the ribosome This process requires: – An elongation factor, EF-Tu – Energy from GTP – The formation of a peptide bond between the amino acids
3-35 Overview of Translation Elongation 3-36 Termination of Translation • Three different codons (UAG, UAA, UGA) cause translation termination • Proteins called release factors (not tRNAs) recognize these stop codons causing – Translation to stop – The release of the polypeptide chain • The initiation codon and termination codon at the ends of the mRNA define an open reading frame (ORF) 3-37 Structural Relationship Between Genes, mRNA and Protein Transcription of DNA does not begin or end at same places as translation
– Transcription begins at the transcription initiation site dependent upon the promoter upstream of the gene – Translation begins at the start codon and ends at a stop codon – Therefore mRNA has a 5’-untranslated region/ 5’-UTR and a 3’-UTR or portions of each end of the transcript that are untranslated 3-38 3.2 Replication • Genes replicate faithfully • The Watson-Crick model for DNA replication assumes that as new strands of DNA are made, they follow the usual base-pairing rules of A with T and G with C • Semiconservative replication produces new DNA with each daughter double helix having one parental strand and one new strand
3-39 Types of Replication Alternative theories of replication are: – Semiconservative: each daughter has 1 parental and 1 new strand – Conservative: 2 parental strands stay together – Dispersive: DNA is fragmented, both new and old DNA coexist in the same strand 3-40 3.3 Mutations • Genes accumulate changes or mutations • Mutation is essential for evolution • If a nucleotide in a gene changes, likely a corresponding change will occur in an amino acid of that gene’s protein product – If a mutation results in a different codon for the same amino acid it is a silent mutation
– Often a new amino acid is structurally similar to the old and the change is conservative 3-41 Sickle Cell Disease • Sickle cell disease is a genetic disorder • The disease results from a single base change in the gene for b-globin – The altered base causes the insertion of an incorrect amino acid into the b-globin protein – The altered protein results in distortion of red blood cells under low-oxygen conditions • This disease illustrates that a change in a gene can cause a corresponding change in the protein product of the gene 3-42 Comparison of Sequences from Normal and Sickle-Cell b-globin • The glutamate codon, GAG, is changed to a valine codon, GUG • Changing the gene by one base pair leads to a
disastrous change in the protein product Molecular Biology Fifth Edition Chapter 4 Molecular Cloning Methods Lecture PowerPoint to accompany Robert F. Weaver Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 4-2 4.1 Gene Cloning • Gene cloning is an indispensable molecular biology technique that allows scientists to produce large quantities of their gene of interest • Gene cloning links eukaryotic genes to small bacterial or phage DNAs and inserting these recombinant molecules into bacterial hosts
• Gene cloning can produce large quantities of these genes in pure form 4-3 The Role of Restriction Endonucleases • Restriction endonucleases, first discovered in the late 1960s in E. coli, are named for preventing invasion by foreign DNA by cutting it into pieces • These enzymes cut at sites within the foreign DNA instead of chewing from the ends • By cutting DNA at specific sites they function as finely honed molecular knives 4-4 Naming Restriction Endonucleases Restriction endonucleases are named using the 1st three letters of their name from the Latin name of their source microorganism Hind III
– First letter is from the genus H from Haemophilus – Next two letters are the 1st two letters of the species name in from influenzae – Sometimes the strain designation is included “d” from strain Rd – If microorganism produces only 1 restriction enzyme, end the name with Roman numeral I Hind I – If more than one restriction enzyme is produced, the others are numbered sequentially II, III, IV, etc. 4-5 Restriction Endonuclease Specificity Restriction endonucleases recognize a specific DNA sequence, cutting ONLY at that sequence – They recognize 4-bp, 6-bp, 8-bp palindromic sequences – The frequency of cuts lessens as the recognition sequence is longer
– They cut DNA reproducibly in the same place 4-6 Restriction-Modification System • What prevents these enzymes from cutting up the host DNA? – They are paired with methylases – Theses enzymes recognize, methylate the same site • Together they are called a restriction-modification system, R-M system • Methylation protects DNA, after replication the parental strand is already methylated 4-7 An Experiment Using Restriction Endonuclease: Boyer and Cohen
• An early experiment used EcoRI to cut 2 plasmids, small circular pieces of DNA independent of the host chromosome • Each plasmid had 1 EcoRI site • Cutting converted circular plasmids into linear DNA with the same sticky ends – The ends base pair • Some ends re-close • Others join the 2 pieces • DNA ligase joins 2 pieces with covalent bonds 4-8 Summary • Restriction endonucleases recognize specific sequences in DNA molecules and make cuts in both strands • This allows very specific cutting of DNAs • The cuts in the two strands are frequently staggered, so restriction enzymes can create sticky ends that help to link together 2 DNAs to
form a recombinant DNA in vitro 4-9 Vectors • Vectors function as DNA carriers to allow replication of recombinant DNAs • Typical experiment uses 1 vector plus a piece of foreign DNA – The inserted and foreign DNA depends on the vector for its replication as it does not have an origin of replication, the site where DNA replication begins • There are 2 major classes of vectors: – Plasmids – Phages 4-10 Plasmids As Vectors • pBR plasmids were developed early but are rarely used today • pUC series is similar to pBR – 40% of the DNA has been deleted – Cloning sites are clustered together into one
area called the multiple cloning site (MCS) – MCS allows one to cut the vector and foreign gene with two different restriction enzymes and use a directional cloning technique to know the orientation of the insert 4-11 Screening: antibiotics and b-galactosidase Screening capabilities within plasmids: – Antibiotic resistance genes (e.g., ampicillin resistance gene) allow for the selection of bacteria that have received a copy of the vector – Multiple cloning site inserted into the gene lacZ’ coding for the enzyme b-galactosidase • Clones with foreign DNA in the MCS disrupt the ability of the cells to make b-galactosidase • Plate on media with a b-galactosidase indicator (X-gal) and clones with intact b-galactosidase enzyme will produce blue colonies • Colorless colonies should contain the plasmid with foreign DNA compared to blue colonies that do not contain the plasmid with DNA
4-12 Summary • First generation plasmid cloning vectors include pBR322 and the pUC plasmids • Screening capabilities: – Ampicillin resistance gene – MCS that interrupts a b-galactosidase gene • MCS facilitates directional cloning into 2 different restriction sites for orientation of inserted gene 4-13 Phages As Vectors • Bacteriophages are natural vectors that transduce bacterial DNA from one cell to another • Phage vectors infect cells much more efficiently than plasmids transform cells • Clones are not colonies of cells using phage vectors, but rather plaques, a clearing of the bacterial lawn due to phage killing the bacteria in that area
4-14 l Phage Vectors • First phage vectors were constructed by Fred Blattner and colleagues – Modifications included removal of the middle region and retention of the genes needed for phage replication – Could replace removed phage genes with foreign DNA • Advantage: Phage vectors can receive larger amounts of foreign DNA (up to 20 kb of DNA) – Traditional plasmid vectors take much less • Phage vectors require a minimum size foreign DNA piece (12 kb) inserted to package into a phage particle 4-15 Cloning Using a Phage Vector 4-16
Genomic Libraries • A genomic library contains clones of all the genes from a species genome • Restriction fragments of a genome can be packaged into phage using about 16 – 20 kb per fragment • This fragment size will include the entirety of most eukaryotic genes • Once a library is established, it can be used to search for any gene of interest 4-17 Selection via Plaque Hybridization • Searching a genomic library requires a probe to determine which clone contains the desired gene • Ideal probe – labeled nucleic acid with sequence matching
the gene of interest 4-18 Cosmids Cosmids are designed for cloning large DNA fragments – Behave both as plasmid and phage and contain • cos sites, cohesive ends of phage DNA that allow the DNA to be packaged into a l phage head • Plasmid origin of replication permitting replication as plasmid in bacteria – Nearly all l genome removed so there is room for large inserts (40-50 kb) – Very little phage DNA yields them unable to replicate, but they are infectious and carry their recombinant DNA into bacterial cells 4-19 M13 Phage Vectors • Long, thin, filamentous phage • Contains:
– Gene fragment with b-galactosidase – Multiple cloning site like the pUC family • Advantage – This phage’s genome is single-stranded DNA – Fragments cloned into it will be recovered in single-stranded form 4-20 M13 Cloning to Recover Single-stranded DNA Product • After infecting E. coli cells, single-stranded phage DNA is converted to double-stranded replicative form (RF) • Use the replicative form for cloning foreign DNA into MCS • Recombinant DNA infects host cells resulting in single-stranded recombinant DNA • Phage particles, containing single-stranded phage DNA is secreted from transformed cells and can be collected from media
4-21 Phagemids Phagemids are also vectors – Like cosmids have aspects of both phages and plasmids – Has MCS inserted into lacZ’ gene to screen blue/ white colonies – Has origin of replication of single-stranded phage f1 to permit recovery of single- stranded recombinant DNA – MCS has 2 phage RNA polymerase promoters, 1 on each side of MCS 4-22 Summary
• Two kinds of phage are popular cloning vectors - l phage - Has nonessential genes removed making room for inserts up to 20kb - Cosmids can accept DNA up to 50 kb - M13 phage - Has MCS - Produces single-stranded recombinant DNA • Plasmids called phagemids also produce single- stranded DNA in presence of helper phage • Engineered phage can accommodate inserts up to 20 kb, useful for building genomic libraries 4-23 Eukaryotic Vectors and Very High Capacity Vectors • There are vectors designed for cloning genes into eukaryotic cells • Other vectors are based on the Ti plasmid to carry genes into plant cells • Yeast artificial chromosomes (YAC) and bacterial artificial chromosomes (BAC) are
used for cloning huge pieces of DNA 4-24 Identifying a Specific Clone With a Specific Probe • Probes are used to identify a desired clone from among the thousands of irrelevant ones • Two types are widely used – Polynucleotides (also called oligonucleotides) – Antibodies 4-25 Polynucleotide Probes Looking for the gene you want, you might use the homologous gene from another organism – If already cloned and there is enough sequence similarity to permit hybridization – Need to lower stringency of hybridization conditions to tolerate some mismatches – High temperature, high organic solvent concentration
and low salt concentration are factors that promote separation of two strands in a DNA double helix and can be adjusted as needed 4-26 Protein-based Polynucleotide Probes No homologous DNA from another organism? • If amino acid sequence is known, deduce a set of nucleotide sequences to code for these amino acids • Construct these nucleotide sequences chemically using the synthetic probes • Why use several? – Genetic code is degenerate with most amino acids having more than 1 nucleic acid triplet – Must construct several different nucleotide sequences for most amino acids 4-27 Summary • Specific clones can be identified using
polynucleotide probes binding to the gene itself • Knowing the amino acid sequence of the gene product permits design of a set of oligonucleotides that encode part of the amino acid sequence • This can be a very quick and accurate means of identifying a particular clone 4-28 cDNA Cloning • cDNA - complementary DNA or copy DNA that is a DNA copy of RNA • A cDNA library is a set of clones representing as many as possible of the mRNAs in a given cell type at a given time – Such a library can contain tens of thousands of different clones 4-29 Making a cDNA Library 4-30
Reverse Transcriptase • Central to successful cloning is the synthesis of cDNA from a mRNA template using reverse transcriptase (RT), an RNA- dependent DNA polymerase – RT cannot initiate DNA synthesis without a primer – Use the poly(A) tail at 3’ end of most eukaryotic mRNA so that oligo(dT) may serve as primer 4-31 Ribonuclease H • RT with oligo(dT) primer has made a single-stranded DNA off of mRNA • Need to remove the RNA • Partially degrade the mRNA using ribonuclease H (RNase H) – Enzyme degrades RNA strand of an RNA- DNA hybrid – Remaining RNA fragments serve as primers for “second strand” DNA using nick translation
4-32 Nick Translation • The nick translation process simultaneously: – Removes DNA ahead of a nick – Synthesizes DNA behind nick – Net result moves the nick in the 5’ to 3’ direction • Enzyme often used is E. coli DNA polymerase I – Has 5’ to 3’ exonuclease activity – Allows enzyme to degrade DNA ahead of the nick 4-33 Terminal Transferase • cDNAs don’t have the sticky ends of genomic DNA cleaved with restriction enzymes • Blunt ends will ligate, but is inefficient • Generate sticky ends using enzyme terminal
deoxynucleotidyl transferase (TdT), terminal transferase with one dNTP – If use dCTP with the enzyme – dCMPs are added one at a time to 3’ ends of the cDNA – Same technique adds oligo(dG) to 5’ ends of the vector – Generate ligation product ready for transformation 4-34 Summary • cDNA library can be synthesized using mRNAs from a cell as templates for the 1st strand that is then used as a template for the 2nd strand – Reverse transcriptase generates 1st strand – DNA polymerase I generates the second strand • Give cDNAs oligonucleotide tails that base-pair with complementary tails on a cloning vector • Use these recombinant DNAs to transform bacteria • Detect clones with: – Colony hybridization using labeled probes – Antibodies if gene product translated
4-35 4.2 The Polymerase Chain Reaction • Polymerase chain reaction (PCR) is used to amplify DNA and can be used to yield a DNA fragment for cloning • Invented by Kary Mullis and colleagues in 1980s • Special heat-stable polymerases are now used that are able to work after high temperatures - researchers no longer need to add fresh DNA polymerase after each round of replication 4-36 Standard PCR • Use enzyme DNA polymerase to copy a selected region of DNA – Add short pieces of DNA (primers) that hybridize to DNA sequences on either side of piece of interest – causes initiation of DNA synthesis through that area, X – Copies of both strands of X and original DNA strands are templates for the next round of DNA
synthesis – The selected region of DNA now doubles in amount with each synthesis cycle 4-37 Amplifying DNA by PCR 4-38 Using Reverse Transcriptase PCR (RT- PCR) in cDNA Cloning • To clone a cDNA from just one mRNA whose sequence is known, a type of PCR called reverse transcriptase PCR (RT-PCR) can be used • Difference between PCR and RT-PCR – Starts with a mRNA, not dsDNA – Begin by converting mRNA to DNA – Use forward primers to convert ssDNA to dsDNA – Continue with standard PCR 4-39
Real-Time PCR • Real-time PCR quantifies the amplification of the DNA as it occurs • As the DNA strands separate, they anneal to forward and reverse primers, and to a fluorescent-tagged oligonucleotide complementary to part of one DNA strand that serves as a reporter probe 4-40 Real-Time PCR • A fluorescent-tagged oligonucleotide serves as a reporter probe – Fluorescent tag at 5’-end – Fluorescence quenching tag at 3’- end • As PCR progresses from the forward primer the 5’ tag is separated from the 3’ tag and allows the 5’ tag to fluoresce • Fluorescence increases with incorporation into DNA product
and can be quantified 4-41 4.3 Methods of Expressing Cloned Genes Cloning a gene permits • Production of large quantities of a particular DNA sequence for detailed study • Large quantities of the gene’s product can also be obtained for further use – Study – Commerce 4-42 Expression Vectors • Vectors discussed so far are used to first put a foreign DNA into a bacterium to replicate and screen • Expression vectors are those that can yield protein products of the cloned genes
– Bacterial expression vectors typically have two elements required for active gene expression; a strong promoter and a ribosome binding site near an initiating codon 4-43 Controlling the lac Promoter • lac promoter is somewhat inducible – Stays off until stimulated by inducer IPTG – However, repression is typically incomplete or leaky and some expression will still occur • To avoid this problem, use a plasmid or phagemid carrying its own lacI repressor gene to keep the cloned gene off until it is induced by IPTG 4-44 Alternative to the lac Promoter • Promoter from ara operon, PBAD, allow fine control of transcription – Inducible by arabinose, a sugar – Transcription rate varies with arabinose
concentration 4-45 Summary • Expression vectors are designed to yield the protein product of a cloned gene • To optimize expression, these vectors include strong bacterial or phage promoters and bacterial ribosome binding sites • Most cloning vectors are inducible, which avoids premature overproduction of a foreign product that could poison the bacterial host cells 4-46 Expression Vectors That Produce Fusion Proteins • Most vectors express fusion proteins – The actual natural product of the gene isn’t made – Extra amino acids help in purifying the protein product
• Oligohistidine expression vector has a short sequence just upstream of MCS encoding 6 His – Oligohistidine has a high affinity for divalent metal ions like nickel (Ni2+) – Permits purification by nickel affinity chromatography – The his tag can be removed using enzyme enterokinase without damage to the protein product 4-47 Using an Oligohistidine Expression Vector 4-48 Expression vector lgt11 • This phage contains the lac control region followed by the lacZ gene • The cloning sites are located within the lacZ gene
• Products of gene correctly inserted will be fusion proteins with a b-galactosidase leader 4-49 Detecting positive lgt11 clones via antibody screening • Lambda phages with cDNA inserts are plated • Protein released are blotted onto a support • Probe with antibody specific to protein • Antibody bound to protein from plaque is detected with labeled protein A 4-50
Summary • Expression vectors frequently produce fusion proteins with one part of the protein coming from the coding sequences in the vector and the other part from sequences in the cloned gene • Many fusion proteins have advantage of being simple to isolate by affinity chromatography • Vector lgt11 produces fusion proteins that can be detected in plaques with a specific antiserum 4-51 Bacterial Expression System Shortcomings • There are problems with expression of eukaryotic proteins in a bacterial system – Bacteria may recognize the proteins as foreign and destroy them – Post-translational modifications are different in bacteria – Bacterial environment may not permit correct protein folding • Very high levels of cloned eukaryotic proteins can be expressed in useless,
insoluble form 4-52 Eukaryotic Expression Systems • Avoid bacterial expression problems by expressing the protein in a eukaryotic cell • Initial cloning done in E. coli using a shuttle vector, able to replicate in both bacterial and eukaryotic cells • Yeast is suited for this purpose – Rapid growth and ease of culture – A eukaryote with more appropriate post- translational modification – Use of the yeast export signal peptide secretes protein into growth medium for easy purification 4-53 Use of Baculovirus As Expression Vector • Viruses in this class have a large circular DNA genome, 130 kb • Major viral structural protein is made in huge amounts in infected cells – The promoter for this protein, polyhedrin, is
very active – These vectors can produce up to 0.5 g of protein per liter of medium – Nonrecombinant viral DNA entering cells does not result in infectious virus as it lacks an essential gene supplied by the vector 4-54 Animal Cell Transfection • Carried out in two ways: • Calcium phosphate – Mix cells with DNA in a phosphate buffer and add a solution of calcium salt to form a precipitate – The cells take up the calcium phosphate crystals, which include some DNA • Liposomes – The DNA is mixed with lipid to form liposomes, small vesicles with some of the DNA inside – DNA-bearing liposomes fuse with the cell membrane to deliver DNA inside the cell 4-55
Summary • Foreign genes can be expressed in eukaryotic cells • These eukaryotic systems have advantages over prokaryotic systems for producing eukaryotic proteins – The proteins tend to fold properly and are soluble, rather than aggregated into insoluble inclusion bodies – Post-translational modifications are compatible 4-56 Using the Ti Plasmid to Transfer Genes to Plants • Genes can be introduced into plants with vectors that can replicate in plant cells • Common bacterial vector promoters and replication origins are not recognized by plant cells • Plasmids are used containing T-DNA – T-DNA is derived from a plasmid known as tumor-inducing (Ti)
– Ti plasmid comes from bacteria that cause plant tumors called crown galls 4-57 Ti Plasmid Infection • Bacterium infects plant, transfers Ti plasmid to host cells • T-DNA integrates into the plant DNA causing abnormal proliferation of plant cells • T-DNA genes direct the synthesis of unusual organic acids, opines which can serve as an energy source to the infecting bacteria but are useless to the plant 4-58 The Ti Plasmid Transfers Crown Gall 4-59 Use of the T-DNA Plasmid 4-60
Summary • Molecular biologists can transfer cloned genes to plants, creating transgenic organisms with altered characteristics, using a plant vector such as the Ti plasmid Molecular Biology Fifth Edition Chapter 5 Molecular Tools for Studying Genes and Gene Activity Lecture PowerPoint to accompany Robert F. Weaver Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5-2 5.1 Molecular Separations • Often mixtures of proteins or nucleic acids are generated during the course of
molecular biological procedures – A protein may need to be purified from a crude cellular extract – A particular nucleic acid molecule made in a reaction needs to be purified • Gel electrophoresis is used to separate different species of: – Nucleic acid – Protein 5-3 DNA Gel Electrophoresis • Melted agarose is poured into a form equipped with removable comb • Comb “teeth” form slots in the solidified agarose • DNA samples are placed in the slots • An electric current is run through the gel at a neutral pH to allow the sample to
travel through the gel matrix 5-4 DNA Separation by Agarose Gel Electrophoresis • DNA is negatively charged due to the phosphates in its backbone and moves toward the positive pole – Small DNA pieces have little frictional drag so they move rapidly – Large DNAs have more frictional drag so their mobility is slower – Distributes DNA according to size • Largest near the top • Smallest near the bottom • DNA is stained with fluorescent dye that intercalates between the bases 5-5 DNA Size Estimation
• Mobility of fragments are plotted v. log of molecular weight (or number of base pairs) • Electrophoresis of unknown DNA in parallel with standard fragments permits size estimation upon comparison • Same principles apply to RNA separation 5-6 Electrophoresis of Large DNA • Special techniques are required for DNA fragments larger than about 1 kilobases • Instead of constant current, alternate long pulses of current in forward direction with shorter pulses in either opposite or sideways direction • Technique is called pulsed-field gel electrophoresis (PFGE)
5-7 Protein Gel Electrophoresis • Separation of proteins is done using polyacrylamide gel electrophoresis (PAGE) – Treat proteins to denature subunits with detergent such as sodium dodecyl sulfate (SDS) • SDS coats polypeptides with negative charges so all move to anode • Masks natural charges of protein subunits so all move relative to mass not charge – As with DNA smaller proteins move faster toward the anode 5-8 Summary • DNAs, RNAs, and proteins of various masses can be separated by gel electrophoresis • Most common gel used in nucleic acid electrophoresis is agarose but polyacrylamide is typically used in protein electrophoresis
• SDS-PAGE is used to separate polypeptides according to their masses 5-9 Two-Dimensional Gel Electrophoresis • While SDS-PAGE gives good resolution of polypeptides, some mixtures are so complex that additional resolution is needed • Two-dimensional gel electrophoresis: – Nondenaturing gel electrophoresis (no SDS) uses 2 consecutive gels each in a different dimension – Sequential gels with distinct pH separation and polyacrylamide gel concentration 5-10 Two-Dimensional Gel Electrophoresis Technique A two process method: • Isoelectric focusing gel: mixture of proteins electrophoresed through gel in a narrow
tube containing a pH gradient – Negatively charged protein moves to its isoelectric point at which it is no longer charged – Tube gel is removed and used as the sample in the second process 5-11 • Standard SDS-PAGE: – Tube gel is removed and used as the sample at the top of a standard polyacrylamide gel – Proteins partially resolved by isoelectric focusing are further resolved according to size • When used to a compare complex mixtures of proteins prepared under two different conditions, even subtle differences are visible Two-Dimensional Gel Electrophoresis Technique continued 5-12 Ion-Exchange Chromatography
• Chromatography originally referred to the pattern seen after separating colored substances on paper • Ion-exchange chromatography uses a resin to separate substances by charge • This is especially useful for proteins • Resin is placed in a column and the sample is loaded onto the column material 5-13 Separation by Ion-Exchange Chromatography • Once the sample is loaded buffer is passed over the resin + sample • As ionic strength of elution buffer increases, samples of solution flowing through the column are collected • Samples are tested for the presence of the protein of interest
5-14 Gel Filtration Chromatography • Protein size is a valuable property that can be used as a basis of physical separation • Gel filtration uses columns filled with porous resins that let in smaller substances and exclude larger substances • As a result larger substances travel faster through the column 5-15 Affinity Chromatography • The resin contains a substance to which the molecule of interest has a strong and specific affinity • The molecule binds to a column resin coupled to the affinity reagent – Molecule of interest is retained – Most other molecules flow through without binding – Last, the molecule of interest is eluted from the column using a specific solution that disrupts their specific binding
5-16 Summary • High-resolution separation of proteins can be achieved by two-dimensional gel electrophoresis • Ion-exchange chromatography can be used to separate substances according to their sizes • Gel filtration chromatography uses columns filled with porous resins that let in smaller substances but exclude larger ones • Affinity chromatography is a powerful purification technique that exploits an affinity reagent with strong and specific affinity for a molecule of interest 5-17 5.2 Labeled Tracers • For many years “labeled” has been synonymous with “radioactive” • Radioactive tracers allow vanishingly small quantities of substances to be detected
• Molecular biology experiments typically require detection of extremely small amounts of a particular substance 5-18 Autoradiography Autoradiography is a means of detecting radioactive compounds with a photographic emulsion – Preferred emulsion is x-ray film – DNA is separated on a gel and radiolabeled – Gel is placed in contact with x- ray film for hours or days – Radioactive emissions from the labeled DNA expose the film – Developed film shows dark bands 5-19 Autoradiography Analysis
• Relative quantity of radioactivity can be assessed looking at the developed film • More precise measurements are made using a densitometer – Area under peaks on a tracing by a scanner – Proportional to darkness of the bands on autoradiogram 5-20 Liquid Scintillation Counting Radioactive emissions from a sample create photons of visible light are detected by a photomultiplier tube in the process of liquid scintillation counting – Remove the radioactive material (band from gel) to a vial containing scintillation fluid – Fluid contains a fluor that fluoresces when hit with radioactive emissions – Acts to convert invisible radioactivity into visible light
5-21 Nonradioactive Tracers • Newer nonradioactive tracers now rival older radioactive tracers in sensitivity • These tracers do not have hazards: – Health exposure – Handling – Disposal • Increased sensitivity is from use of a multiplier effect of an enzyme that is coupled to probe for molecule of interest 5-22 Detecting Nucleic Acids With a Nonradioactive Probe 5-23 5.3 Using Nucleic Acid Hybridization • Hybridization is the ability of one single- stranded nucleic acid to form a double helix with another single strand of
complementary base sequence • Previous discussion focused on colony and plaque hybridization • This section looks at techniques for isolated nucleic acids 5-24 Southern Blots: Identifying Specific DNA Fragments • Digests of genomic DNA are separated on a gel • The separated pieces are transferred to filter (nitrocellulose) by diffusion, or more recently by electrophoresing the DNA onto the filter • The filter is then treated with alkali to denature the DNA, resulting ssDNA binds to the filter • A labeled cDNA probe that is complementary to the DNA of interest is then applied to the filter • A positive band should be detectable where hybridization between the probe and DNA occurred 5-25
Southern Blots • The probe hybridizes and a band is generated corresponding to the DNA fragment of interest • Visualize bands with x-ray film or autoradiography • Multiple bands can lead to several interpretations – Multiple genes – Several restriction sites in the gene 5-26 DNA Fingerprinting and DNA Typing • Southern blots are used in forensic labs to identify individuals from DNA-containing materials • Minisatellite DNA is a sequence of bases repeated several times, also called a DNA fingerprint – Individuals differ in the pattern of repeats of the basic sequence
– The difference is large enough that 2 people have only a remote chance of having exactly the same pattern of repeats 5-27 DNA Fingerprinting Process is a Southern blot • Cut the DNA under study with restriction enzyme – Ideally cut on either side of minisatellite but not inside • Run the digested DNA on a gel and blot • Probe with labeled minisatellite DNA and image – Note that real samples result in very complex patterns 5-28 Forensic Uses of DNA Fingerprinting
and DNA Typing • While people have different DNA fingerprints, parts of the pattern are inherited in a Mendelian fashion – Can be used to establish parentage – Potential to identify criminals – Remove innocent people from suspicion • Actual pattern has so many bands they can smear together indistinguishably – Forensics uses probes for just a single locus – Set of probes gives a set of simple patterns 5-29 In Situ Hybridization: Locating Genes in Chromosomes • Labeled probes can be used to hybridize to chromosomes and reveal which chromosome contains the gene of interest – Spread chromosomes from a cell – Partially denature DNA creating single-stranded regions to hybridize to labeled probe – Stain chromosomes and detect presence of label on particular chromosome • Probe can be detected with a fluorescent
antibody in a technique called fluorescence in situ hybridization (FISH) 5-30 Immunoblots Immunoblots (also called Western blots) use a similar process to Southern blots – Electrophoresis of proteins – Blot the proteins from the gel to a membrane – Detect the protein using antibody or antiserum to the target protein – Labeled secondary antibody is used to bind the first antibody for visualization and to increase the signal 5-31 Summary • Labeled DNA (or RNA) probes can be used to hybridize to DNAs of the same or very similar sequence on a Southern blot • DNA fingerprinting can be used as a forensic tool or to test parentage • In situ hybridization can be used to locate genes
or other specific DNA sequences on whole chromosomes • Proteins can be detected and quantified in a complex mixture using Western blots 5-32 5.4 DNA Sequencing • Sanger, Maxam, and Gilbert developed 2 methods for determining the exact base sequence of a cloned piece of DNA • Modern DNA sequencing is based on the Sanger method and uses dideoxy nucleotides to terminate DNA synthesis – The process yields a series of DNA fragments whose size is measured by electrophoresis – The last base in each fragment is known as that dideoxy nucleotide was used to terminate the reaction – Ordering the fragments by size tells the base sequence of the DNA
5-33 Sanger Method of DNA Sequencing 5-34 Automated DNA Sequencing • Manual sequencing is powerful but slow • Automated sequencing uses dideoxynucleotides tagged with different fluorescent molecules – Products of each dideoxynucleotide will fluoresce a different color – Four reactions are completed, then mixed together and run out on one lane of a gel 5-35 High Throughput Sequencing • Once an organism’s genome sequence is known, very rapid sequencing techniques can be applied
to sequence the genome of another member of the same species • Produces relatively short reads or contiguous sequences (25-35bp or 200-300bp, depending on the method) that can easily be pieced together if a reference sequence is available 5-36 High Throughput Sequencing • Pyrosequencing is one example that is an automated system with the advantages of speed and accuracy - nucleotides are added one by one and the incorporation of a nucleotide is detected by a release of pyrophosphate, which leads to a flash of light • Another method (Illumina company) starts by attaching short pieces of DNA to a solid surface, amplifying each DNA in a tiny patch on the surface, then sequencing the patches together by extending them one nucleotide at a time using fluorescent chain-terminating nucleotides, whose fluoresce reveals their identity
5-37 Restriction Mapping • Prior to the start of large-scale sequencing preliminary work is done to locate landmarks – A map based on physical characteristics is called a physical map – If restriction sites are the only map features then a restriction map has been prepared 5-38 Restriction Map Example • Consider a 1.6 kb piece of DNA as an example • Cut separate samples of the original 1.6 kb fragment with different restriction enzymes • Separate the digests on an agarose gel to determine the size of pieces from each digest • Can also use same digest to
find the orientation of an insert cloned into a vector 5-39 Southern Blots and Restriction Mapping 5-40 Summary • Physical maps tell about the spatial arrangement of physical “landmarks” such as restriction sites – In restriction mapping cut the DNA in question with 2 or more restriction enzymes in separate reactions – Measure the sizes of the resulting fragments – Cut each with another restriction enzyme and measure size of subfragments by gel electrophoresis • Sizes permit location of some restriction sites relative to others • Improve process by Southern blotting fragments and hybridizing them to labeled fragments from another restriction enzyme to reveal overlaps
5-41 5.5 Protein Engineering With Cloned Genes: Site-Directed Mutagenesis • Cloned genes permit biochemical microsurgery on proteins – Specific bases in a gene may be changed – Amino acids at specific sites in the protein product may be altered as a result – Effects of those changes on protein function can be observed 5-42 PCR-based Site-Directed Mutagenesis 5-43 Summary • Using cloned genes, one can introduce changes that may alter the amino acid sequence of the corresponding protein products • Mutagenized DNA can be made with: – Double-stranded DNA – Two complementary mutagenic primers
– PCR • Digest the PCR product to remove wild-type DNA • Cells can be transformed with mutagenized DNA 5-44 5.6 Mapping and Quantifying Transcripts • In the field of molecular biology mapping (locating start and end) and quantifying (how much transcript exists at a set time) transcripts are common procedures • Often transcripts do not have a uniform terminator, resulting in a continuum of species smeared on a gel • Techniques that are specific for the sequence of interest are important 5-45 Northern Blots • Northern blots detect RNA • Example: You have cloned a cDNA – Question: How actively is the corresponding gene expressed in different tissues?
– Answer: Find out using a Northern Blot • Obtain RNA from different tissues • Run RNA on agarose gel and blot to membrane • Hybridize to a labeled cDNA probe – Northern plot tells abundance of the transcript – Quantify using densitometer 5-46 S1 Mapping Use S1 mapping to locate the ends of RNAs and to determine the amount of a given RNA in cells at a given time – Label a ssDNA probe that can only hybridize to transcript of interest – Probe must span the sequence start to finish – After hybridization, treat with S1 nuclease which degrades ssDNA and RNA – Transcript protects part of the probe from degradation – Size of protected area can be measured by gel electrophoresis 5-47
Summary • A Northern blot is similar to a Southern blot but is a method used for detection of RNA • In S1 mapping, a labeled DNA probe is used to detect 5’- or 3’-end of a transcript • Amount of probe protected is proportional to concentration of transcript, so S1 mapping can be quantitative • RNase mapping is a variation on SI mapping that uses an RNA probe and RNase 5-48 Run-Off Transcription • A good assay to measure the rate of in vitro transcription • DNA fragment containing gene to transcribe is cut with restriction enzyme in middle of transcription region • Transcribe the truncated fragment in vitro using labeled nucleotides, as polymerase
reaches truncation it “runs off” the end • Measure length of run-off transcript compared to location of restriction site at 3’- end of truncated gene 5-49 Summary • Run-off transcription is a means of checking efficiency and accuracy of in vitro transcription – Gene is truncated in the middle and transcribed in vitro in presence of labeled nucleotides – RNA polymerase runs off the end making an incomplete transcript – Size of run-off transcript locates transcription start site – Amount of transcript reflects efficiency of transcription 5-50 5.7 Measuring Transcription Rates in Vivo • Primer extension, S1 mapping and Northern blotting will determine the concentration of specific transcripts at a
given time • These techniques do not really reveal the rate of transcript synthesis as concentration involves both: – Transcript synthesis – Transcript degradation 5-51 Reporter Gene Transcription • Place a surrogate reporter gene under the control of a specific promoter and measure the accumulation of the product of this reporter gene • The reporter genes are carefully chosen to have products very convenient to assay – lacZ produces b-galactosidase which has a blue cleavage product – cat produces chloramphenicol acetyl transferase (CAT) which inhibits bacterial growth – Luciferase produces a chemiluminescent compound that emits light 5-52
Measuring Protein Accumulation in Vivo • Gene activity can be monitored by measuring the accumulation of protein, the ultimate gene product • There are two primary methods of measuring protein accumulation – Immunoblotting / Western blotting (discussed earlier) – Immunoprecipitation • Immunoprecipitation typically uses an antibody that will bind specifically to the protein of interest followed with a secondary antibody complexed to Protein A on resin beads using a low-speed centrifuge 5-53 5.8 Assaying DNA-Protein Interactions • Study of DNA-protein interactions is of significant interest to molecular biologists • Types of interactions often studied: – Protein-DNA binding – Which bases of DNA interact with a protein 5-54
Filter Binding Filter binding is used to measure DNA- protein interaction and based on the fact that double-stranded DNA will not bind by itself to a filter, but a protein-DNA complex will – Double-stranded DNA can be labeled and mixed with protein – Assay protein-DNA binding by measuring the amount of label retained on the filter 5-55 Nitrocellulose Filter-Binding Assay • dsDNA is labeled and mixed with protein • Pour dsDNA through a nitrocellulose filter • Measure amount of radioactivity that passed through filter and retained on filter 5-56 Gel Mobility Shift • DNA moves through a gel faster when it is not
bound to protein • Gel shift assays detect interaction between protein and DNA by reduction of the electrophoretic mobility of a small DNA bound to a protein 5-57 Footprinting • Footprinting detects protein-DNA interaction and will show where a target lies on DNA and which bases are involved in protein binding • Three methods are very popular: – DNase footprinting – Dimethylsulfate footprinting – Hydroxyl radical footprinting 5-58 DNase Footprinting Protein binding to DNA covers the binding site and protects from attack by DNase
• End label DNA, 1 strand only • Protein binds DNA • Treat complex with DNase I mild conditions for average of 1 cut per molecule • Remove protein from DNA, separate strands and run on a high-resolution polyacrylamide gel 5-59 Summary • Footprinting finds target DNA sequence or binding site of a DNA-binding protein • DNase footprinting binds protein to end-labeled DNA target, then attacks DNA-protein complex with DNase • DNA fragments are electrophoresed with protein binding site appearing as a gap in the pattern where protein protected DNA from degradation
5-60 Chromatin Immunoprecipitation (ChIP) • ChIP is a method used to discover whether a given protein is bound to a given gene in chromatin - the DNA-protein complex that is the natural state of the DNA in a living cell • ChIP uses an antibody to precipitate a particular protein in complex with DNA, and PCR to determine whether the protein binds near a particular gene 5-61 Chromatin Immunoprecipitation (ChIP) 5-62 5.9 Assaying Protein-Protein Interactions • Immunoprecipitation uses an antibody that will bind specifically to the protein of interest and, using a low-speed centrifuge, will ‘pull-down’ any proteins associated with the protein of interest
5-63 5.11 Knockouts and Transgenes • Probing structures and activities of genes does not answer questions about the role of the gene in the life of the organism • Targeted disruption of genes is now possible in several organisms • When genes are disrupted in mice the products are called knockout mice • Foreign genes, called transgenes, can also be added to an organism, such as a mouse, to create transgenic mice 5-64 Knockout Results • Phenotype may not be obvious in the progeny, but still instructive • Other cases can be lethal with the mice dying before birth • Intermediate effects are also common and may require monitoring during the life of
the mouse 5-65 Methods to Generate Transgenic Mice • Two methods to generate transgenic mice: • 1. Injection of cloned foreign gene into the sperm pronucleus just after fertilization of a mouse egg but before the sperm and egg nuclei have fused to allow for insertion of the foreign DNA into the embryonic cell DNA • 2. Injection of cloned foreign DNA into mouse embryonic stem cells, creating transgenic ES cells • Both methods produce chimeric mice that must undergo several rounds of breeding and selection to find true transgenic animals 5-66 Summary • To probe the role of a gene, molecular
biologists can perform targeted disruption of the corresponding gene in a mouse and then look for the effects of the mutation in the ‘knockout mouse’ or insert the foreign gene as a transgene in the ‘transgenic mouse’ Molecular Biology Fifth Edition Chapter 6 The Mechanism of Transcription in Bacteria Lecture PowerPoint to accompany Robert F. Weaver Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 6-2 6.1 RNA Polymerase Structure By 1969 SDS-PAGE of RNA polymerase from E. coli had shown several subunits
– 2 very large subunits are b (150 kD) and b’ (160 kD) – Sigma (s) at 70 kD – Alpha (a) at 40 kD – 2 copies present in holoenzyme – Omega (ω) at 10 kD • Was not clearly visible in SDS-PAGE, but seen in other experiments • Not required for cell viability or in vivo enzyme activity • Appears to play a role in enzyme assembly 6-3 Sigma as a Specificity Factor • Core enzyme without the s subunit could not transcribe viral DNA, yet had no problems with highly nicked calf thymus DNA • With s subunit, the holoenzyme worked equally well on both types of DNA 6-4
Summary • The key player in the transcription process is RNA polymerase • The E. coli enzyme is composed of a core, which contains the basic transcription machinery, and a s-factor, which directs the core to transcribe specific genes 6-5 6.2 Promoters • Why was the core RNA polymerase capable of transcribing nicked DNA in the previous table? • Nicks and gaps are good sites for RNA polymerase to bind nonspecifically • The presence of the s-subunit permits recognition of authentic RNA polymerase binding sites called promoters • Transcription that begins at promoters is specific, directed by the s-subunit 6-6 Binding of RNA Polymerase to Promoters
• How tightly does core enzyme v. holoenzyme bind DNA? • Experiment measures binding of DNA to enzyme using nitrocellulose filters – Holoenzyme binds filters tightly – Core enzyme binding is more transient 6-7 Temperature and RNA Polymerase Binding • As the temperature is lowered, the binding of RNA polymerase to DNA decreases dramatically • Higher temperatures promote DNA melting and encourage RNA
polymerase binding 6-8 RNA Polymerase Binding Hinkle and Chamberlin proposed: • RNA polymerase holoenzyme binds DNA loosely at first – Binds at promoter initially – Scans along the DNA until it finds a promoter • Complex with holoenzyme loosely bound at the promoter is a closed promoter complex as DNA is in a closed ds form • Holoenzyme can then melt a short DNA region at the promoter to form an open promoter complex with polymerase bound tightly to DNA 6-9 Polymerase/Promoter Binding • Holoenzyme binds DNA loosely at first
• Complex loosely bound at promoter = closed promoter complex, dsDNA in closed form • Holoenzyme melts DNA at promoter forming open promoter complex - polymerase tightly bound 6-10 Summary • The s-factor allows initiation of transcription by causing the RNA polymerase holoenzyme to bind tightly to a promoter • This tight binding depends on local melting of the DNA to form an open promoter complex and is stimulated by s • The s-factor can therefore select which genes will be transcribed 6-11
Core Promoter Elements • There is a region common to bacterial promoters described as 6-7 bp centered about 10 bp upstream of the start of transcription = -10 box • Another short sequence centered 35 bp upstream is known as the -35 box • Comparison of thousands of promoters has produced a consensus sequence (or most common sequence) for each of these boxes 6-12 Promoter Strength • Consensus sequences: – -10 box sequence approximates TATAAT – -35 box sequence approximates TTGACA • Mutations that weaken promoter binding: – Down mutations – Increase deviation from the consensus sequence • Mutations that strengthen promoter binding: – Up mutations – Decrease deviation from the consensus sequence
6-13 UP Element • The UP element is upstream of the core promoter, stimulating transcription by a factor of 30 • UP is associated with 3 “Fis” sites which are binding sites for the transcription- activator protein Fis, not for the polymerase itself 6-14 The rrnB P1 Promoter • Transcription from the rrn promoters respond –Positively to increased concentration of iNTP –Negatively to the alarmone ppGpp 6-15 6.3 Transcription Initiation • Transcription initiation was assumed to end as RNA polymerase formed 1st phosphodiester bond
• Carpousis and Gralla found that very small oligonucleotides (2-6 nt long) are made without RNA polymerase leaving the DNA • Abortive transcripts such as these have been found up to 10 nt 6-16 Stages of Transcription Initiation • Formation of a closed promoter complex • Conversion of the closed promoter complex to an open promoter complex • Polymerizing the early nucleotides – polymerase at the promoter • Promoter clearance – transcript becomes long enough to form a stable hybrid with template 6-17 Reuse of s
• During initiation s can be recycled for additional use with a new core polymerase • The core enzyme can release s which is then free to associate with another core enzyme 6-18 Models for the s-Cycle • The obligate release version of the s-cycle model arose from experiments performed by Travers and Burgess that proposed the dissociation of s from core as polymerase undergoes promoter clearance and switches from initiation to elongation mode • The stochastic release model proposes that s is indeed released from the core polymerase but that there is no discrete point of release during transcription and that the release occurs at random - a preponderance of evidence favors this model
6-19 Local DNA Melting at the Promoter • From the number of RNA polymerase holoenzymes bound to DNA, it was calculated that each polymerase caused a separation of about 10 bp • In another experiment, the length of the melted region was found to be 12 bp • Later, size of the DNA transcription bubble in complexes where transcription was active was found to be 17-18 bp 6-20 Promoter Clearance • RNA polymerases have evolved to recognize and bind strongly to promoters • This poses a challenge when it comes time for promoter clearance as those strong bonds must be broken in order for polymerase to leave the promoter and enter the elongation phase 6-21 Promoter Clearance
• Several hypotheses have been proposed • The polymerase cannot move enough downstream to make a 10-nt transcript without doing one of three things: - transient excursion: moving briefly downstream and then snapping back to the starting position - inchworming: stretching itself by leaving its trailing edge in place while moving its leading edge downstream - scrunching: compressing the DNA without moving itself 6-22 Abortive Transcription, Scrunching and Promoter Clearance • Ebert and colleagues performed several experiments to distinguish between the hypotheses • Using E.coli polymerase the authors concluded that approximately 100% of all transcription cycles involved scrunching, which suggested that scrunching is required for promoter clearance • The E.coli polymerase achieves abortive transcription by scrunching: drawing downstream
DNA into the polymerase without actually moving and losing its grip on promoter DNA • The scrunched DNA could store enough energy to allow the polymerase to break its bonds to the promoter and begin productive transcription 6-23 Structure and Function of s • Genes encoding a variety of s-factors have been cloned and sequenced • There are striking similarities in amino acid sequence clustered in 4 regions • Conservation of sequence in these regions suggests important function • All of the 4 sequences are involved in binding to core and DNA 6-24 Homologous Regions in Bacterial s Factors 6-25 E. coli s70
• Four regions of high sequence similarity are indicated • Specific areas that recognize the core promoter elements are the -10 box and – 35 box 6-26 Region 1 • Role of region 1 appears to be in preventing s from binding to DNA by itself • This is important as s binding to promoters could inhibit holoenzyme binding and thereby inhibit transcription Region 2 • This region is the most highly conserved of the four • There are four subregions – 2.1 to 2.4 • 2.4 recognizes the promoter’s -10 box • The 2.4 region appears to be a-helix 6-27 Regions 3 and 4 • Region 3 is involved in both core and
DNA binding • Region 4 is divided into 2 subregions – This region seems to have a key role in promoter recognition – Subregion 4.2 contains a helix-turn-helix DNA-binding domain and appears to govern binding to the -35 box of the promoter 6-28 Summary • Comparison of different s gene sequences reveals 4 regions of similarity among a wide variety of sources • Subregions 2.4 and 4.2 are involved in promoter -10 box and -35 box recognition • The s-factor by itself cannot bind to DNA, but DNA interaction with core unmasks a DNA- binding region of s • Region between amino acids 262 and 309 of b’ stimulates s binding to the nontemplate strand in the -10 region of the promoter
6-29 Role of a-Subunit in UP Element Recognition • RNA polymerase itself can recognize an upstream promoter element, UP element • While s-factor recognizes the core promoter elements, what recognizes the UP element? • It appears to be the a-subunit of the core polymerase 6-30 Modeling the Function of the C- Terminal Domain • RNA polymerase binds to a core promoter via its s- factor, no help from C- terminal domain of a-subunit • Binds to a promoter with an UP element using s plus the a-subunit C-terminal domains (CTD) • Results in very strong
interaction between polymerase and promoter • This produces a high level of transcription 6-31 6.4 Elongation • After transcription initiation is accomplished, core polymerase continues to elongate the RNA • Nucleotides are added sequentially, one after another in the process of elongation 6-32 Function of the Core Polymerase • Core polymerase contains the RNA synthesizing machinery • Phosphodiester bond formation involves the b- and b’-subunits • These subunits also participate in DNA binding • Assembly of the core polymerase is a
major role of the a-subunit 6-33 Role of b in Phosphodiester Bond Formation • Core subunit b lies near the active site of the RNA polymerase • This active site is where the phosphodiester bonds are formed linking the nucleotides • The s-factor may also be near the nucleotide-binding site during the initiation phase 6-34 Structure of the Elongation Complex • This section will examine how well predictions have been borne out by structural studies • How does the polymerase deal with problems of unwinding and rewinding templates? • How does it move along the helical template without twisting RNA product
around the template? 6-35 RNA-DNA Hybrid • The area of RNA-DNA hybridization within the E. coli elongation complex extends from position –1 to –8 or –9 relative to the 3’ end of the nascent RNA • In T7 the similar hybrid appears to be 8 bp long 6-36 Structure of the Core Polymerase • X-ray crystallography on the Thermus aquaticus RNA polymerase core reveals an enzyme shaped like a crab claw • It appears designed to grasp the DNA • A channel through the enzyme includes the catalytic center – Mg2+ ion coordinated by 3 Asp residues – Rifampicin-binding site
6-37 Structure of the Holoenzyme-DNA Complex Crystal structure of T. aquaticus holoenzyme-DNA complex as an open promoter complex reveals: – DNA is bound mainly to s-subunit – Interactions between amino acids in region 2.4 of s and -10 box of promoter are possible – 3 highly conserved aromatic amino acids are able to participate in promoter melting as predicted – 2 invariant basic amino acids in s predicted to function in DNA binding are positioned to do so – A form of the polymerase that has 2 Mg2+ ions 6-38 Structure of the Elongation Complex • The X-ray crystal structure of the Thermus thermophilus RNA polymerase elongation complex in 2007 revealed several important observations – a valine residue in the E’ subunit inserts into the minor groove of the downstream DNA – the downstream DNA is double-stranded up
to and including the +2 base pair – the enzyme can accommodate nine base pairs of RNA-DNA hybrid – the RNA product in the exit channel is twisted into the shape it would assume as 1/2 of an A-form dsRNA 6-39 Topology of Elongation • Elongation of transcription involves polymerization of nucleotides as the RNA polymerase travels along the template DNA • Polymerase maintains a short melted region of template DNA • DNA must unwind ahead of the advancing polymerase and rewind behind it • Strain introduced into the template DNA ahead of the transcription bubble is relaxed by topoisomerases 6-40 Pausing and Proofreading
• RNA polymerase frequently pauses, or even backtracks, during elongation • Pausing allows ribosomes to keep pace with the RNA polymerase, and it is the first step in termination • Backtracking aids proofreading by extruding the 3’-end of the RNA out of the polymerase, where misincorporated nucleotides can be removed by an inherent nuclease activity of the polymerase, stimulated by auxiliary factors 6-41 6.5 Termination of Transcription • When the polymerase reaches a terminator at the end of a gene it falls off the template and releases the RNA • There are 2 main types of terminators – Intrinsic terminators function with the RNA polymerase by itself without help from other proteins – Other type depends on auxiliary factor called -dependent
terminators 6-42 Rho-Independent Termination • Intrinsic or rho-independent termination depends on terminators of 2 elements: – Inverted repeats followed immediately by – T-rich region in the nontemplate strand of the gene • An inverted repeat predisposes a transcript to form a hairpin structure due to complementary base pairing between the inverted repeat sequences 6-43 Inverted Repeats and Hairpins • The repeat at right is symmetrical around its center shown with a dot • A transcript of this sequence is self-
complementary – Bases can pair up to form a hairpin as seen in the lower panel 6-44 Model of Intrinsic Termination Bacterial terminators act by: • Base-pairing of something to the transcript to destabilize RNA-DNA hybrid – Causes hairpin to form • This causes transcription to pause – a string of U’s incorporated just downstream of hairpin to destabilize the hybrid and the RNA falls off the DNA template 6-45
Rho-Dependent Termination • Rho caused depression of the ability of RNA polymerase to transcribe phage DNAs in vitro • This depression was due to termination of transcription • After termination, polymerase must reinitiate to begin transcribing again 6-46 Rho Affects Chain Elongation • There is little effect of rho or r on transcription initiation, if anything it is increased • The effect of rho or r on total RNA synthesis is a significant decrease • This is consistent with action of rho or r to terminate transcription forcing time- consuming reinitiation 6-47 Rho Causes Production of Shorter Transcripts
• Synthesis of much smaller RNAs occurs in the presence of rho or r compared to those made in the absence • To ensure that this due to r itself and not to RNase activity of r, RNA was transcribed without r and then incubated in the presence of r • There was no loss of transcript size, so no RNase activity in r 6-48 Rho Releases Transcripts from the DNA Template • Compare the sedimentation of transcripts made in presence and absence of r – Without r, transcripts cosedimented with the DNA template – they hadn’t been released – With r present in the incubation, transcripts sedimented more slowly – they were not associated with the DNA template • It appears that r serves to release the RNA transcripts from the DNA template 6-49
Mechanism of Rho • No string of T’s in the r- dependent terminator, just inverted repeat to hairpin • Binding to the growing transcript, r follows the RNA polymerase • It catches the polymerase as it pauses at the hairpin • Releases transcript from the DNA-polymerase complex by unwinding the RNA-DNA hybrid 6-50 Summary • Using the trp attenuator as a model rho-independet terminator revealed two important features: 1 - an inverted repeat that allows a hairpin to for at the end of the transcript 2 - a string of T’s in the nontemplate strand that results in a string of weak rU-dA base pairs holding the transcript to the template strand
• Rho-dependent terminators consist of an inverted repeat, which can cause a hairpin to form in the transcript but no string of T’s Molecular Biology Fifth Edition Chapter 7 Operons: Fine Control of Bacterial Transcription Lecture PowerPoint to accompany Robert F. Weaver Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 7-2 7.1 The lac Operon • The lac operon was the first operon
discovered • It contains 3 genes coding for E. coli proteins that permit the bacteria to use the sugar lactose – Galactoside permease (lacY) which transports lactose into the cells -galactosidase (lacZ) cuts the lactose into galactose and glucose – Galactoside transacetylase (lacA) whose function is unclear 7-3 Genes of the lac Operon • The genes are grouped together: – lacZ = b-galactosidase – lacY = galactoside permease – lacA = galactoside transacetylase • All 3 genes are transcribed together producing 1 mRNA, a polycistronic message that starts from a single promoter – Each cistron, or gene, has its own ribosome binding site – Each cistron can be translated by separate ribosomes that bind independently of each other
7-4 Control of the lac Operon • The lac operon is tightly controlled, using 2 types of control – Negative control, like the brake of a car, must remove the repressor from the operator - the “brake” is a protein called the lac repressor – Positive control, like the accelerator pedal of a car, an activator, additional positive factor responds to low glucose by stimulating transcription of the lac operon 7-5 Negative Control of the lac Operon • Negative control indicates that the operon is turned on unless something intervenes and stops it • The off-regulation is done by the lac repressor – Product of the lacI gene
– Tetramer of 4 identical polypeptides – Binds the operator just right of promoter 7-6 lac Repressor • When the repressor binds to the operator, the operon is repressed – Operator and promoter sequence are contiguous – Repressor bound to operator prevents RNA polymerase from binding to the promoter and transcribing the operon • As long as no lactose is available, the lac operon is repressed 7-7 Negative Control of the lac Operon 7-8 Inducer of the lac Operon • The repressor is an allosteric protein – Binding of one molecule to the protein changes
shape of a remote site on that protein – Altering its interaction with a second molecule • The inducer binds the repressor – Causing the repressor to change conformation that favors release from the operator • The inducer is allolactose, an alternative form of lactose 7-9 Inducer of the lac Operon • The inducer of the lac operon binds the repressor • The inducer is allolactose, an alternative form of lactose 7-10 Discovery of the Operon During the 1940s and 1950s, Jacob and Monod studied the metabolism of lactose by E. coli •Three enzyme activities / three genes were induced together by galactosides
• Constitutive mutants need no induction, genes are active all the time • Created merodiploids or partial diploid bacteria carrying both wild-type (inducible) and constitutive alleles 7-11 Discovery of the Operon • Using merodiploids or partial diploid bacteria carrying both wild-type and constitutive alleles distinctions could be made by determining whether the mutation was dominant or recessive • Because the repressor gene produces a repressor protein that can diffuse throughout the nucleus, it can bind to both operators in a meriploid and is called a trans-acting gene because it can act on loci on both DNA molecules • Because an operator controls only the operon on the same DNA molecule it is called a cis-acting gene
7-12 Effects of Regulatory Mutations: Wild-type and Mutated Repressor 7-13 Effects of Regulatory Mutations: Wild-type and Mutated Operator with Defective Binding 7-14 Repressor-Operator Interactions • Using a filter-binding assay, the lac repressor binds to the lac operator • A genetically defined constitutive lac operator has lower than normal affinity for the lac repressor • Sites defined by two methods as the operator are in fact the same 7-15 The Mechanism of Repression
• The repressor does not block access by RNA polymerase to the lac promoter • Polymerase and repressor can bind together to the lac promoter • Polymerase-promoter complex is in equilibrium with free polymerase and promoter 7-16 lac Repressor and Dissociation of RNA Polymerase from lac Promoter • Without competitor, dissociated polymerase returns to promoter • Heparin and repressor prevent reassociation of polymerase and promoter • Repressor prevents reassociation by binding to the operator adjacent to the promoter
Molecular Biology Fifth Edition Chapter 1 A Brief Hi.docx
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Molecular Biology Fifth Edition Chapter 1 A Brief Hi.docx

  • 1. Molecular Biology Fifth Edition Chapter 1 A Brief History Lecture PowerPoint to accompany Robert F. Weaver Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 1-2 A Brief History • What is molecular biology? – The attempt to understand biological phenomena in molecular terms – The study of gene structure and function at the molecular level • Molecular biology is a melding of aspects of genetics and biochemistry
  • 2. 1-3 1.1 Transmission Genetics • Transmission genetics deals with the transmission of traits from parental organisms to their offspring • The chemical composition of genes was not known until 1944 – Gene - genetic units – Phenotype - observable characteristics 1-4 Mendel’s Laws of Inheritance • A gene can exist in different forms called alleles • One allele can be dominant over the other, recessive, allele • The first filial generation (F1) contains offspring of the original parents • If each parent carries two copies of a gene, the parents are diploid for that gene
  • 3. 1-5 Mendel’s Laws of Inheritance • Homoozygotes have two copies of the same allele • Heterozygotes have one copy of each allele • Parents in 1st mating are homozygotes, having 2 copies of one allele • Sex cells, or gametes, are haploid, containing only 1 copy of each gene • Heterozygotes produce gametes having either allele • Homozygotes produce gametes having only one allele 1-6 Summary • Genes can exist in several different forms or alleles • One allele can be dominant over the other, so heterozygotes having two different alleles of one gene will generally exhibit the characteristic dictated by the dominant allele • The recessive allele is not lost; it can still exert its influence when paired with another recessive allele
  • 4. in a homozygote 1-7 The Chromosome Theory of Inheritance • Chromosomes are discrete physical entities that carry genes • Thomas Hunt Morgan used the fruit fly, Drosophila melanogaster, to study genetics • Autosomes occur in pairs in a given individual (not the X or the Y chromosome) • Sex chromosomes are identified as X and Y – Females have two X chromosomes – Males have one X and one Y chromosome 1-8 Location of Genes on a Chromosome • Every gene has its place, or locus, on a chromosome • Genotype is the combination of alleles found in an organism • Phenotype is the visible expression of the genotype – Wild-type phenotype is the most common or
  • 5. generally accepted standard – Mutant alleles are usually recessive 1-9 Genetic Recombination and Mapping • In early experiments genes on separate chromosomes behaved independently • Genes on the same chromosome behaved as if they were linked • This genetic linkage is not absolute • Offspring show new combinations of alleles not seen in the parents when recombination occurs 1-10 Recombination • During meiosis, gamete formation, crossing over can occur resulting in the exchange of genes between the two homologous chromosomes • The result of the crossing-over event produces a new combination of alleles
  • 6. • This process is called recombination 1-11 Genetic Mapping • Morgan proposed that the farther apart two genes are on a chromosome, the more likely they are to recombine • If two loci recombine with a frequency of 1%, they are said to be separated by a map distance of one centimorgan (named for Morgan) • This mapping observation applies both to prokaryotes and to eukaryotes 1-12 Physical Evidence for Recombination • Microscopic examination of the maize chromosome provided direct physical observation of recombination using easily identifiable features of one chromosome • Similar observations were made in Drosophila • Recombination was detected both
  • 7. physically and genetically in both animals and plants 1-13 Summary • The chromosome theory of inheritance holds that genes are arranged in linear fashion on chromosomes • Certain traits tend to be inherited together when the genes for those traits are on the same chromosome • Recombination between two homologous chromosomes during meiosis can scramble the parental alleles to yield nonparental combinations • The farther apart two genes are on a chromosome the more likely it is that recombination will occur 1-14 1.2 Molecular Genetics • The Discovery of DNA: The general structure of nucleic acids was discovered by the end of the 19th century
  • 8. – Long polymers or chains of nucleotides – Nucleotides are linked by sugars through phosphate groups • Composition of Genes: DNA? RNA? Protein? In 1944, Avery and his colleagues demonstrated that genes are composed of DNA 1-15 The Relationship between Genes and Proteins • Experiments have shown that a defective gene gives a defective or absent enzyme • This lead to the proposal that one gene is responsible for making one enzyme • Proposal not quite correct for 3 reasons: 1. One enzyme may be composed of several polypeptides, each gene codes for only one polypeptide 2. Many genes code for non-enzyme proteins 3. End products of some genes are not polypeptides (i.e. tRNA, rRNA)
  • 9. 1-16 Activities of Genes Genes perform three major roles • Replicated faithfully • Direct the production of RNAs and proteins • Accumulate mutations thereby allowing for evolution 1-17 Replication • Franklin and Wilkins produced x-ray diffraction data on DNA, Watson and Crick proposed that DNA is double helix – Two DNA strands wound around each other – Strands are complementary – if you know the sequence of one strand, you automatically know the sequence of the other strand • Semiconservative replication keeps one strand of the parental double helix conserved in each of the daughter double helices
  • 10. 1-18 Genes Direct the Production of Polypeptides • Gene expression is the process by which a gene product is made • Two steps are required – 1. Transcription: DNA is transcribed into RNA – 2. Translation: the mRNA is read or translated to assemble a protein – Codon: a sequence of 3 nucleic acid bases that code for one amino acid within the mRNA 1-19 Genes Accumulate Mutations Genes change in several ways • Change one base to another • Deletions of one base up to a large segment • Insertions of one base up to a large segment
  • 11. • The more drastic the change, the more likely it is that the gene or genes involved will be totally inactivated 1-20 Summary • All cellular genes are made of DNA arranged in a double helix • This structure explains how genes replicate, carry information and collect mutations • The sequence of nucleotides in a gene is a genetic code that carries the information for making an RNA • A change in the sequence of bases constitutes and mutation, which can change the sequence of amino acids in the genes polypeptide product 1-21 1.3 The Three Domains of Life Current research theories support the division of living organisms into three domains 1. Bacteria
  • 12. 2. Eukaryota 3. Archaea • Like bacteria as they are organisms without nuclei • More similar to eukaryotes in the context of their molecular biology 1-22 Archaea Archaea live in the most inhospitable regions of the earth • Thermophiles tolerate extremely high temperatures • Halophiles tolerate very high salt concentrations • Methanogens produce methane as a by-product of metabolism Molecular Biology Fifth Edition Chapter 2 The Molecular Nature
  • 13. of Genes Lecture PowerPoint to accompany Robert F. Weaver Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 2-2 The Nature of Genetic Material Historical Background • Miescher isolated nuclei from pus (white blood cells) in 1869 – Found a novel phosphorus-bearing substance = nuclein – Nuclein is mostly chromatin, a complex of DNA and chromosomal proteins • End of 19th century – DNA and RNA separated from proteins • Levene, Jacobs, et al. characterized basic composition of DNA and RNA 2-3
  • 14. Molecular Foundation: Early experiments that explored the question: What is the genetic material? • Key experiments performed by Frederick Griffith in 1928 • Observed change in Streptococcus pneumoniae — from avirulent (R) rough colonies, bacteria without capsules, to virulent (S) smooth colonies, bacteria that had capsules • Result: Heat-killed virulent bacteria could transform avirulent bacteria into virulent bacteria 2-4 Outline of Griffith’s Transformation Experiments 2-5 DNA: The Transforming Material In 1944 Avery, Macleod and McCarty used a transformation test similar to Griffith’s procedure taking care to define the chemical
  • 15. nature of the transforming substance – Techniques used excluded both protein and RNA as the chemical agent of transformation – Exclusion of DNA verified that DNA is the chemical agent of transformation of S. pneumoniae from avirulent to virulent 2-6 Analytical Tools Physical-chemical analysis has often used: 1. Ultracentrifugation Used to estimate size of material 2. Electrophoresis Indicated high charge-to-mass ratio 3. Ultraviolet Absorption Spectrophotometry Absorbance of UV light matched that of DNA 4. Elementary Chemical Analysis Nitrogen-to-phosphorus ratio of 1.67, expected for DNA but lower than expected for protein 2-7
  • 16. Confirmation for DNA as the genetic material • In the 1940s geneticists doubted the use of DNA as the genetic material as it appeared to be monotonous repeats of 4 bases • By 1953 Watson & Crick published the double- helical model of DNA structure and Chargaff demonstrated that the 4 bases were not present in equal proportions • In 1952 Hershey and Chase demonstrated that bacteriophage infection comes from DNA, adding more evidence to support that DNA is the genetic material 2-8 Outline of Hershey and Chase’s Experiment 2-9 Summary • The classic molecular biology experiments performed by Griffith, Avery, MacLeod,
  • 17. Mccarty, Hershey and Chase combined revealed that DNA is the genetic element 2-10 The Chemical Nature of Polynucleotides • Biochemists determined the components of nucleotides during the 1940s • The component parts of DNA – Nitrogenous bases: • Adenine (A) • Cytosine (C) • Guanine (G) • Thymine (T) – Phosphoric acid – Deoxyribose sugar 2-11 Nucleosides and Deoxyribose • RNA component parts – Nitrogenous bases • Like DNA except Uracil
  • 18. (U) replaces Thymine – Phosphoric acid – Ribose sugar • Bases use ordinary numbers • Carbons in sugars are noted as primed numbers • Nucleotides contain phosphoric acid • Nucleosides lack the phosphoric acid • Deoxyribose lacks a hydroxyl group (OH) at the 2-position 2-12 Purines and Pyrimidines • Adenine and guanine are related structurally to the parent molecule purine • Cytosine, thymine and uracil resemble the parent molecule pyrimidine 2-13
  • 19. DNA Linkage • Nucleotides are nucleosides with a phosphate group attached through a phosphodiester bond • Nucleotides may contain one, two, or even three phosphate groups linked in a chain 2-14 A Trinucleotide The example trinucleotide has polarity – The top of molecule has a free 5’-phosphate group = 5’ end – The bottom has a free 3’- hydroxyl group = 3’ end 2-15 Summary
  • 20. • DNA and RNA are chain-like molecules composed of subunits called nucleotides • Nucleotides contain a base linked to the 1’-position of a sugar and a phosphate group • The phosphate joins the sugars in a DNA or RNA chain through their 5’- and 3’- hydroxyl groups by phosphodiester bonds 2-16 DNA Structure The Double Helix • Rosalind Franklin’s x-ray diffraction data suggested that DNA had a helical shape • The data also indicated a regular, repeating structure • Chargaff’s data revealed that the content of purines was always roughly equal to pyrimidines • Watson and Crick proposed a double helix with sugar-phosphate backbones on the outside and bases aligned on the interior 2-17
  • 21. DNA Helix • Structure compared to a twisted ladder – Curving sides of the ladder represent the sugar- phosphate backbone – Ladder rungs are the base pairs – There are about 10 base pairs per turn • Arrows indicate that the two strands are antiparallel 2-18 Summary • The DNA molecule is a double helix, with sugar-phosphate backbones on the outside and base pairs on the inside • The bases pair in a specific way: – Adenine (A) with thymine (T) – Guanine (G) with cytosine (C) 2-19
  • 22. Genes Made of RNA Viruses are a package of genes – No metabolic activity of their own – When a virus infects a host cell, the cellular machinery is diverted and begins to make viral proteins – Viral genes are replicated and used for the production of viral protein that assemble into virus particles Viruses contain nucleic acid, some viruses use DNA genes, but some viruses have RNA genes, either double- or single-stranded 2-20 Physical Chemistry of Nucleic Acids DNA and RNA molecules can appear in several different structural variants – Changes in relative humidity will cause variation in DNA molecular structure – The twist of the DNA molecule is normally shown to be right-handed, but left-handed DNA also exists and was identified in 1979
  • 23. 2-21 A Variety of DNA Structures • High humidity (92%) DNA is called the B-form • Reduce relative humidity to 75% and DNA takes on the A-form – Plane of base pairs in A- form is no longer perpendicular to the helical axis – The A-form is seen when one strand of DNA is hybridized with one strand of RNA strand • When wound in a left- handed helix, DNA is found in the Z-form • To date at least one gene requires Z-DNA for activation
  • 24. 2-22 Summary • In the cell, DNA may exist in the common B form, with horizontal base pairs • A very small fraction of the DNA may assume a left-handed helical form called the Z-form • An RNA-DNA hybrid assumes a third helical shape, called the A-form, with base pairs tilted away from the horizontal 2-23 Variation in DNA between Organisms • Ratios of G to C and A to T are fixed in any specific organism • The total percentage of G + C varies over a range of 22 to 73% • These reflect differences in physical
  • 25. properties 2-24 DNA Denaturation or Melting • With heating, noncovalent forces holding DNA strands together weaken and break • When the forces break, the two strands come apart in denaturation or melting • The temperature at which the DNA strands are ½ denatured is the melting temperature or Tm • GC content of DNA has a significant effect on Tm with higher GC content yielding a higher Tm 2-25 DNA Denaturation • In addition to heat, DNA can be denatured by: – Organic solvents – High pH – Low salt concentration • GC content also affects DNA density
  • 26. – Direct, linear relationship – Due to larger molar volume of A-T base pairs compared to G-C base pairs 2-26 Summary • The GC content of a natural DNA can vary from less than 25% to almost 75% • The GC content has a strong effect on the physical properties of the DNA, each of which increase linearly with GC content – The melting temperature, the temperature at which the two strands are half-dissociated or denatured – Density – Low ionic strength, high pH and organic solvents also promote DNA denaturation 2-27 DNA Renaturation
  • 27. • After two DNA strands separate, under proper conditions the strands can come back together • Process is called annealing or renaturation • Three most important factors: – Temperature – best at about 25 C below Tm – DNA Concentration – within limits higher concentration better likelihood that 2 complementary will find each other – Renaturation Time – as increase time, more annealing will occur 2-28 Polynucleotide Chain Hybridization Hybridization is a process of putting together a combination of two different nucleic acids – Strands could be 1 DNA and 1 RNA – Also could be 2 DNA with complementary or nearly
  • 28. complementary sequences 2-29 DNA Sizes DNA size is expressed in 3 different ways: – Number of base pairs – Molecular weight – 660 is molecular weight of 1 base pair – Length – 33.2 Å per helical turn of 10.4 base pairs DNA can be measured by electron microscopy or gel electrophoresis 2-30 DNAs of Various Sizes and Shapes • Bacterial DNA is typically circular • Some DNA will be linear • Supercoiled DNA coils or wraps around itself like a twisted rubber band
  • 29. 2-31 Summary • Natural DNAs come in sizes ranging from several kilobases to thousands of megabases • The size of a small DNA can be estimated by electron microscopy • This technique can also reveal whether a DNA is circular or linear and whether it is supercoiled 2-32 Relationship between DNA Size and Genetic Capacity How does one know how many genes are in a particular piece of DNA? – Can’t determine from DNA size alone – Factors include: • How much of the DNA is devoted to genes? • What is the space between genes? – One can estimate the upper limit of number genes a piece of DNA can hold
  • 30. 2-33 DNA Size and Genetic Capacity How many genes are in a piece of DNA? – Start with basic assumptions • Genes encode protein (ignoring the RNAs made) • The average protein is abut 40,000 D – How many amino acids does this represent? • Average mass of an amino acid is about 110 D • Average protein – 40,000 / 110 = 364 amino acids • Each amino acid = 3 DNA base pairs • 364 amino acids requires 1092 base pairs 2-34 DNA Genetic Capacity How large is an average piece of DNA? – E. coli chromosome • 4.6 x 106 bp • ~4200 proteins – Phage l (infects E. coli) • 4.85 x 104 bp • ~44 proteins – • 5375 bp • ~5 proteins (squeezes in more by overlapping
  • 31. genes) 2-35 DNA Content and the C-Value Paradox • The C-value is the DNA content per haploid cell • One might expect that more complex organisms need more genes than simple organisms • For the mouse or human compared to yeast this is correct • Yet the frog has 7 times more genes per cell than humans 2-36 C-Value Paradox • The observation that more complex organisms will not always need more genes than simple organisms is called the C-value paradox • The most likely explanation for the paradox is that organisms with extraordinarily high C-values simply have
  • 32. a great deal of extra, noncoding DNA 2-37 Summary • There is a rough correlation between DNA content and number of genes in a cell or virus • This correlation breaks down in several cases of closely related organisms where the DNA content per haploid cell (C-value) varies widely • The C-value paradox is probably explained not by extra genes, but by extra noncoding DNA in some organisms Molecular Biology Fifth Edition Chapter 3 An Introduction to Gene Function Lecture PowerPoint to accompany Robert F. Weaver
  • 33. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 3-2 3.1 Storing Information Producing a protein from DNA involves both transcription and translation – A codon is the 3 base sequence that determines what amino acid is used – Template strand is the DNA strand that is used to generate the mRNA – Nontemplate strand is not used in transcription 3-3 Protein Structure Proteins are chain-like polymers of small subunits, called amino acids
  • 34. – DNA has 4 different nucleotides (A,G, C, T) – Proteins have 20 different amino acids with: • An amino group • A hydroxyl group • A hydrogen atom • A specific side chain 3-4 Polypeptides • Amino acids are joined together via peptide bonds • Chains of amino acids are called polypeptides • Proteins are composed of 1 or more polypeptides • Polypeptides have polarity – Free amino group at one end is the amino- or N-terminus – Free hydroxyl group at the other end is the carboxyl- or C-terminus 3-5 Types of Protein Structure (4) • The linear order of amino acids is a protein’s primary structure • Interaction of the amino acids’ amino and carboxyl groups gives rise to the secondary structure of a protein – Secondary structure is the result of amino acid and
  • 35. carboxyl group hydrogen bonding among near neighbors – Common types of secondary structure: -helix -sheet 3-6 Helical Secondary Structure • In a-helix secondary structure polypeptide backbone groups H bond with each other • The dashed lines indicate hydrogen bonds between nearby amino acids 3-7 Sheet Secondary Structure • The b-sheet pattern of 2° structure also occurs when
  • 36. polypeptide backbone groups form H bonds • In the sheet configuration, extended polypeptide chains are packed side by side • This side-by-side packing creates a sheet appearance 3-8 Tertiary Structure • The total three- dimensional shape of a polypeptide is its tertiary structure • A prominent aspect of this structure is the interaction of the amino acid side chains • The globular form of a polypeptide is a roughly
  • 37. spherical structure 3-9 Protein Domains • Compact structural regions of a protein are referred to as domains • Immunoglobulins provide an example of 4 globular domains • Domains may contain common structural-functional motifs – Zinc finger – Hydrophobic pocket • Quaternary structure is the interaction of 2 or more polypeptides 3-10 Summary • Proteins are polymers of amino acids linked through peptide bonds
  • 38. • The sequence of amino acids in a polypeptide (primary structure) gives rise to that molecule’s: – Local shape (secondary structure) – Overall shape (tertiary structure) – Interaction with other polypeptides (quaternary structure) 3-11 Protein Function Proteins: – Provide the structure that help give cells integrity and shape – Serve as hormones carrying signals from one cell to another – Bind and carry substances – Control the activities of genes – Serve as enzymes that catalyze hundreds of chemical reactions 3-12 Relationship Between Genes and Proteins
  • 39. • 1902 Dr. Garrod suggested a link between a human disease and a recessive gene • If a single gene controlled the production of an enzyme, lack of that enzyme could result in the buildup of homogentisic acid which is excreted in the urine • Should the gene responsible for the enzyme be defective, then the enzyme would likely also be defective 3-13 One-Gene/One-Polypeptide • Over time many experiments (i.e., Beadle and Tatum) have built on Garrod’s initial work • Many enzymes contain more than one polypeptide chain and each polypeptide is usually encoded in one gene • These observations have lead to the one gene one polypeptide hypothesis: Most genes contain the information for making one polypeptide 3-14 Information Carrier
  • 40. • In the 1950s and 1960s, the concept that messenger RNA carries information from gene to ribosome was developed • An intermediate carrier was needed as DNA is found in the nucleus, while proteins are made in the cytoplasm • Therefore, some type of molecule must move the information from the DNA in the nucleus to the site of protein synthesis in the cytoplasm 3-15 Discovery of Messenger RNA • Ribosomes are the cytoplasmic site of protein synthesis • Jacob and colleagues proposed that messengers, an alternative of non- specialized ribosomes, translate unstable RNAs • These messengers are independent RNAs that move information from genes to ribosomes 3-16
  • 41. Summary Messenger RNAs carry the genetic information from the genes to the ribosomes, which then synthesize polypeptides 3-17 Transcription • Transcription follows the same base- pairing rules as DNA replication – Remember U replaces T in RNA – This base-pairing pattern ensures that the RNA transcript is a faithful copy of the gene • For transcription to occur at a significant rate, its reaction is enzyme mediated • The enzyme directing transcription is called RNA polymerase 3-18 Synthesis of RNA 3-19
  • 42. Phases of Transcription Transcription occurs in three phases: 1. Initiation 2. Elongation 3. Termination 3-20 Initiation • RNA polymerase recognizes a specific region, the promoter, which lies just upstream of gene • The polymerase binds tightly to the promoter causing localized separation of the two DNA strands • The polymerase starts building the RNA chain by adding ribonucleotides • After several ribonucleotides are joined together the enzyme leaves the promoter and elongation begins
  • 43. 3-21 Elongation • RNA polymerase directs the addition of ribonucleotides in the 5’ to 3’ direction • Movement of the polymerase along the DNA template causes the “bubble” of separated DNA strands to move also • As the RNA polymerase proceeds along the DNA, the two DNA strands that have opened for the “bubble” reform the double helix behind the transciptional machinery 3-22 Transcription and DNA Replication Two fundamental differences between transcription and DNA replication 1. RNA polymerase only makes one RNA strand during transcription, it copies only one DNA strand in a given gene – This makes transcription asymmetrical – Replication is semiconservative 2. DNA melting is limited and transient during
  • 44. transcription, but the separation is permanent in replication 3-23 Termination • Analogous to the initiating activity of promoters, there are regions at the other end of genes that serve to terminate transcription • These terminators work with the RNA polymerase to loosen the association between the RNA product and the DNA template • As a result, the RNA dissociates from the RNA polymerase and the DNA and transcription stops 3-24 Important Note about Conventions • RNA sequences are written 5’ to 3’, left to right • Translation occurs 5’ to 3’ with ribosomes reading the message 5’ to 3’ • Genes are written so that transcription proceeds in
  • 45. a left to right direction • The gene’s promoter area lies just before the start area, said to be upstream of transcription • Genes are therefore said to lie downstream of their promoters 3-25 Summary • Transcription takes place in three stages: – Initiation – Elongation – Termination • Initiation involves the binding of RNA polymerase to the promoter, local melting and forming the first few phosphodiester bonds • During elongation, the RNA polymerase links together ribonucleotides in the 5’ to 3’ direction to make the rest of the RNA • In termination, the polymerase and RNA product dissociate from the DNA template 3-26 Translation - Ribosomes
  • 46. • Ribosomes are protein synthesizing machines – Ribosome subunits are designated with numbers such as 50S or 30S – Number is the sedimentation coefficient - a measure of speed with which the particles sediment through a solution spun in an ultracentrifuge based on mass and shape • Each ribosomal subunit contains RNA and protein 3-27 Ribosomal RNA • The two ribosomal subunits both contain ribosomal RNA (rRNA) molecules and a variety of proteins • rRNAs participate in protein synthesis but do NOT code for proteins • No translation of rRNA occurs 3-28 Summary
  • 47. • Ribosomes are the cell’s protein factories • Bacteria contain 70S ribosomes • Each ribosome has 2 subunits – 50 S – 30 S • Each subunit contains rRNA and many proteins 3-29 tRNA: Translation Adapter Molecule • Generating protein from ribosomes requires change from the nucleic acid to amino acid • This change is described as translation from the nucleic acid base pair language to the amino acid language • Crick proposed that some type of adapter molecule was needed to provide the bridge for translation, perhaps a small RNA • The physical interface between the mRNA and the ribosome
  • 48. 3-30 Transfer RNA: Adapter Molecule • Transfer RNA is a small RNA that recognizes both RNA and amino acids • A cloverleaf model is used to illustrate tRNA structure • The 3’ end binds to a specific amino acid • The anticodon loop contains a 3 nucleotide sequence that pairs with complementarity to a codon in mRNA 3-31 Codons and Anticodons • Enzymes that catalyze attachment of amino acid to tRNA are aminoacyl- tRNA synthetases • A triplet in mRNA is called
  • 49. a codon • The complementary sequence to a codon found in a tRNA is the anticodon 3-32 Summary • Two important sites on tRNAs allow them to recognize both amino acids and nucleic acids • One site binds covalently to an amino acid • The site contains an anticodon that base- pairs with a codon in the mRNA • tRNAs are capable of serving the adapter role and are the key to the mechanism of translation 3-33 Initiation of Protein Synthesis • The initiation codon (AUG) interacts with a special aminoacyl-tRNA
  • 50. – In eukaryotes this is methionyl-tRNA – In bacteria it is a derivative called N-formylmethionyl- tRNA • Position of the AUG codon: – At start of message AUG is initiator – In middle of message AUG is regular methionine • Shine-Dalgarno sequence lies just upstream of the AUG, functions to attract ribosomes – Unique to bacteria – Eukaryotes have special cap on 5’-end of mRNA 3-34 Translation Elongation • During initiation the initiating aminoacyl-tRNA binds within the P site of the ribosome • Elongation adds amino acids one at a time to the initiating amino acid • The first elongation step is binding second aminoacyl-tRNA to the A site on the ribosome This process requires: – An elongation factor, EF-Tu – Energy from GTP – The formation of a peptide bond between the amino acids
  • 51. 3-35 Overview of Translation Elongation 3-36 Termination of Translation • Three different codons (UAG, UAA, UGA) cause translation termination • Proteins called release factors (not tRNAs) recognize these stop codons causing – Translation to stop – The release of the polypeptide chain • The initiation codon and termination codon at the ends of the mRNA define an open reading frame (ORF) 3-37 Structural Relationship Between Genes, mRNA and Protein Transcription of DNA does not begin or end at same places as translation
  • 52. – Transcription begins at the transcription initiation site dependent upon the promoter upstream of the gene – Translation begins at the start codon and ends at a stop codon – Therefore mRNA has a 5’-untranslated region/ 5’-UTR and a 3’-UTR or portions of each end of the transcript that are untranslated 3-38 3.2 Replication • Genes replicate faithfully • The Watson-Crick model for DNA replication assumes that as new strands of DNA are made, they follow the usual base-pairing rules of A with T and G with C • Semiconservative replication produces new DNA with each daughter double helix having one parental strand and one new strand
  • 53. 3-39 Types of Replication Alternative theories of replication are: – Semiconservative: each daughter has 1 parental and 1 new strand – Conservative: 2 parental strands stay together – Dispersive: DNA is fragmented, both new and old DNA coexist in the same strand 3-40 3.3 Mutations • Genes accumulate changes or mutations • Mutation is essential for evolution • If a nucleotide in a gene changes, likely a corresponding change will occur in an amino acid of that gene’s protein product – If a mutation results in a different codon for the same amino acid it is a silent mutation
  • 54. – Often a new amino acid is structurally similar to the old and the change is conservative 3-41 Sickle Cell Disease • Sickle cell disease is a genetic disorder • The disease results from a single base change in the gene for b-globin – The altered base causes the insertion of an incorrect amino acid into the b-globin protein – The altered protein results in distortion of red blood cells under low-oxygen conditions • This disease illustrates that a change in a gene can cause a corresponding change in the protein product of the gene 3-42 Comparison of Sequences from Normal and Sickle-Cell b-globin • The glutamate codon, GAG, is changed to a valine codon, GUG • Changing the gene by one base pair leads to a
  • 55. disastrous change in the protein product Molecular Biology Fifth Edition Chapter 4 Molecular Cloning Methods Lecture PowerPoint to accompany Robert F. Weaver Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 4-2 4.1 Gene Cloning • Gene cloning is an indispensable molecular biology technique that allows scientists to produce large quantities of their gene of interest • Gene cloning links eukaryotic genes to small bacterial or phage DNAs and inserting these recombinant molecules into bacterial hosts
  • 56. • Gene cloning can produce large quantities of these genes in pure form 4-3 The Role of Restriction Endonucleases • Restriction endonucleases, first discovered in the late 1960s in E. coli, are named for preventing invasion by foreign DNA by cutting it into pieces • These enzymes cut at sites within the foreign DNA instead of chewing from the ends • By cutting DNA at specific sites they function as finely honed molecular knives 4-4 Naming Restriction Endonucleases Restriction endonucleases are named using the 1st three letters of their name from the Latin name of their source microorganism Hind III
  • 57. – First letter is from the genus H from Haemophilus – Next two letters are the 1st two letters of the species name in from influenzae – Sometimes the strain designation is included “d” from strain Rd – If microorganism produces only 1 restriction enzyme, end the name with Roman numeral I Hind I – If more than one restriction enzyme is produced, the others are numbered sequentially II, III, IV, etc. 4-5 Restriction Endonuclease Specificity Restriction endonucleases recognize a specific DNA sequence, cutting ONLY at that sequence – They recognize 4-bp, 6-bp, 8-bp palindromic sequences – The frequency of cuts lessens as the recognition sequence is longer
  • 58. – They cut DNA reproducibly in the same place 4-6 Restriction-Modification System • What prevents these enzymes from cutting up the host DNA? – They are paired with methylases – Theses enzymes recognize, methylate the same site • Together they are called a restriction-modification system, R-M system • Methylation protects DNA, after replication the parental strand is already methylated 4-7 An Experiment Using Restriction Endonuclease: Boyer and Cohen
  • 59. • An early experiment used EcoRI to cut 2 plasmids, small circular pieces of DNA independent of the host chromosome • Each plasmid had 1 EcoRI site • Cutting converted circular plasmids into linear DNA with the same sticky ends – The ends base pair • Some ends re-close • Others join the 2 pieces • DNA ligase joins 2 pieces with covalent bonds 4-8 Summary • Restriction endonucleases recognize specific sequences in DNA molecules and make cuts in both strands • This allows very specific cutting of DNAs • The cuts in the two strands are frequently staggered, so restriction enzymes can create sticky ends that help to link together 2 DNAs to
  • 60. form a recombinant DNA in vitro 4-9 Vectors • Vectors function as DNA carriers to allow replication of recombinant DNAs • Typical experiment uses 1 vector plus a piece of foreign DNA – The inserted and foreign DNA depends on the vector for its replication as it does not have an origin of replication, the site where DNA replication begins • There are 2 major classes of vectors: – Plasmids – Phages 4-10 Plasmids As Vectors • pBR plasmids were developed early but are rarely used today • pUC series is similar to pBR – 40% of the DNA has been deleted – Cloning sites are clustered together into one
  • 61. area called the multiple cloning site (MCS) – MCS allows one to cut the vector and foreign gene with two different restriction enzymes and use a directional cloning technique to know the orientation of the insert 4-11 Screening: antibiotics and b-galactosidase Screening capabilities within plasmids: – Antibiotic resistance genes (e.g., ampicillin resistance gene) allow for the selection of bacteria that have received a copy of the vector – Multiple cloning site inserted into the gene lacZ’ coding for the enzyme b-galactosidase • Clones with foreign DNA in the MCS disrupt the ability of the cells to make b-galactosidase • Plate on media with a b-galactosidase indicator (X-gal) and clones with intact b-galactosidase enzyme will produce blue colonies • Colorless colonies should contain the plasmid with foreign DNA compared to blue colonies that do not contain the plasmid with DNA
  • 62. 4-12 Summary • First generation plasmid cloning vectors include pBR322 and the pUC plasmids • Screening capabilities: – Ampicillin resistance gene – MCS that interrupts a b-galactosidase gene • MCS facilitates directional cloning into 2 different restriction sites for orientation of inserted gene 4-13 Phages As Vectors • Bacteriophages are natural vectors that transduce bacterial DNA from one cell to another • Phage vectors infect cells much more efficiently than plasmids transform cells • Clones are not colonies of cells using phage vectors, but rather plaques, a clearing of the bacterial lawn due to phage killing the bacteria in that area
  • 63. 4-14 l Phage Vectors • First phage vectors were constructed by Fred Blattner and colleagues – Modifications included removal of the middle region and retention of the genes needed for phage replication – Could replace removed phage genes with foreign DNA • Advantage: Phage vectors can receive larger amounts of foreign DNA (up to 20 kb of DNA) – Traditional plasmid vectors take much less • Phage vectors require a minimum size foreign DNA piece (12 kb) inserted to package into a phage particle 4-15 Cloning Using a Phage Vector 4-16
  • 64. Genomic Libraries • A genomic library contains clones of all the genes from a species genome • Restriction fragments of a genome can be packaged into phage using about 16 – 20 kb per fragment • This fragment size will include the entirety of most eukaryotic genes • Once a library is established, it can be used to search for any gene of interest 4-17 Selection via Plaque Hybridization • Searching a genomic library requires a probe to determine which clone contains the desired gene • Ideal probe – labeled nucleic acid with sequence matching
  • 65. the gene of interest 4-18 Cosmids Cosmids are designed for cloning large DNA fragments – Behave both as plasmid and phage and contain • cos sites, cohesive ends of phage DNA that allow the DNA to be packaged into a l phage head • Plasmid origin of replication permitting replication as plasmid in bacteria – Nearly all l genome removed so there is room for large inserts (40-50 kb) – Very little phage DNA yields them unable to replicate, but they are infectious and carry their recombinant DNA into bacterial cells 4-19 M13 Phage Vectors • Long, thin, filamentous phage • Contains:
  • 66. – Gene fragment with b-galactosidase – Multiple cloning site like the pUC family • Advantage – This phage’s genome is single-stranded DNA – Fragments cloned into it will be recovered in single-stranded form 4-20 M13 Cloning to Recover Single-stranded DNA Product • After infecting E. coli cells, single-stranded phage DNA is converted to double-stranded replicative form (RF) • Use the replicative form for cloning foreign DNA into MCS • Recombinant DNA infects host cells resulting in single-stranded recombinant DNA • Phage particles, containing single-stranded phage DNA is secreted from transformed cells and can be collected from media
  • 67. 4-21 Phagemids Phagemids are also vectors – Like cosmids have aspects of both phages and plasmids – Has MCS inserted into lacZ’ gene to screen blue/ white colonies – Has origin of replication of single-stranded phage f1 to permit recovery of single- stranded recombinant DNA – MCS has 2 phage RNA polymerase promoters, 1 on each side of MCS 4-22 Summary
  • 68. • Two kinds of phage are popular cloning vectors - l phage - Has nonessential genes removed making room for inserts up to 20kb - Cosmids can accept DNA up to 50 kb - M13 phage - Has MCS - Produces single-stranded recombinant DNA • Plasmids called phagemids also produce single- stranded DNA in presence of helper phage • Engineered phage can accommodate inserts up to 20 kb, useful for building genomic libraries 4-23 Eukaryotic Vectors and Very High Capacity Vectors • There are vectors designed for cloning genes into eukaryotic cells • Other vectors are based on the Ti plasmid to carry genes into plant cells • Yeast artificial chromosomes (YAC) and bacterial artificial chromosomes (BAC) are
  • 69. used for cloning huge pieces of DNA 4-24 Identifying a Specific Clone With a Specific Probe • Probes are used to identify a desired clone from among the thousands of irrelevant ones • Two types are widely used – Polynucleotides (also called oligonucleotides) – Antibodies 4-25 Polynucleotide Probes Looking for the gene you want, you might use the homologous gene from another organism – If already cloned and there is enough sequence similarity to permit hybridization – Need to lower stringency of hybridization conditions to tolerate some mismatches – High temperature, high organic solvent concentration
  • 70. and low salt concentration are factors that promote separation of two strands in a DNA double helix and can be adjusted as needed 4-26 Protein-based Polynucleotide Probes No homologous DNA from another organism? • If amino acid sequence is known, deduce a set of nucleotide sequences to code for these amino acids • Construct these nucleotide sequences chemically using the synthetic probes • Why use several? – Genetic code is degenerate with most amino acids having more than 1 nucleic acid triplet – Must construct several different nucleotide sequences for most amino acids 4-27 Summary • Specific clones can be identified using
  • 71. polynucleotide probes binding to the gene itself • Knowing the amino acid sequence of the gene product permits design of a set of oligonucleotides that encode part of the amino acid sequence • This can be a very quick and accurate means of identifying a particular clone 4-28 cDNA Cloning • cDNA - complementary DNA or copy DNA that is a DNA copy of RNA • A cDNA library is a set of clones representing as many as possible of the mRNAs in a given cell type at a given time – Such a library can contain tens of thousands of different clones 4-29 Making a cDNA Library 4-30
  • 72. Reverse Transcriptase • Central to successful cloning is the synthesis of cDNA from a mRNA template using reverse transcriptase (RT), an RNA- dependent DNA polymerase – RT cannot initiate DNA synthesis without a primer – Use the poly(A) tail at 3’ end of most eukaryotic mRNA so that oligo(dT) may serve as primer 4-31 Ribonuclease H • RT with oligo(dT) primer has made a single-stranded DNA off of mRNA • Need to remove the RNA • Partially degrade the mRNA using ribonuclease H (RNase H) – Enzyme degrades RNA strand of an RNA- DNA hybrid – Remaining RNA fragments serve as primers for “second strand” DNA using nick translation
  • 73. 4-32 Nick Translation • The nick translation process simultaneously: – Removes DNA ahead of a nick – Synthesizes DNA behind nick – Net result moves the nick in the 5’ to 3’ direction • Enzyme often used is E. coli DNA polymerase I – Has 5’ to 3’ exonuclease activity – Allows enzyme to degrade DNA ahead of the nick 4-33 Terminal Transferase • cDNAs don’t have the sticky ends of genomic DNA cleaved with restriction enzymes • Blunt ends will ligate, but is inefficient • Generate sticky ends using enzyme terminal
  • 74. deoxynucleotidyl transferase (TdT), terminal transferase with one dNTP – If use dCTP with the enzyme – dCMPs are added one at a time to 3’ ends of the cDNA – Same technique adds oligo(dG) to 5’ ends of the vector – Generate ligation product ready for transformation 4-34 Summary • cDNA library can be synthesized using mRNAs from a cell as templates for the 1st strand that is then used as a template for the 2nd strand – Reverse transcriptase generates 1st strand – DNA polymerase I generates the second strand • Give cDNAs oligonucleotide tails that base-pair with complementary tails on a cloning vector • Use these recombinant DNAs to transform bacteria • Detect clones with: – Colony hybridization using labeled probes – Antibodies if gene product translated
  • 75. 4-35 4.2 The Polymerase Chain Reaction • Polymerase chain reaction (PCR) is used to amplify DNA and can be used to yield a DNA fragment for cloning • Invented by Kary Mullis and colleagues in 1980s • Special heat-stable polymerases are now used that are able to work after high temperatures - researchers no longer need to add fresh DNA polymerase after each round of replication 4-36 Standard PCR • Use enzyme DNA polymerase to copy a selected region of DNA – Add short pieces of DNA (primers) that hybridize to DNA sequences on either side of piece of interest – causes initiation of DNA synthesis through that area, X – Copies of both strands of X and original DNA strands are templates for the next round of DNA
  • 76. synthesis – The selected region of DNA now doubles in amount with each synthesis cycle 4-37 Amplifying DNA by PCR 4-38 Using Reverse Transcriptase PCR (RT- PCR) in cDNA Cloning • To clone a cDNA from just one mRNA whose sequence is known, a type of PCR called reverse transcriptase PCR (RT-PCR) can be used • Difference between PCR and RT-PCR – Starts with a mRNA, not dsDNA – Begin by converting mRNA to DNA – Use forward primers to convert ssDNA to dsDNA – Continue with standard PCR 4-39
  • 77. Real-Time PCR • Real-time PCR quantifies the amplification of the DNA as it occurs • As the DNA strands separate, they anneal to forward and reverse primers, and to a fluorescent-tagged oligonucleotide complementary to part of one DNA strand that serves as a reporter probe 4-40 Real-Time PCR • A fluorescent-tagged oligonucleotide serves as a reporter probe – Fluorescent tag at 5’-end – Fluorescence quenching tag at 3’- end • As PCR progresses from the forward primer the 5’ tag is separated from the 3’ tag and allows the 5’ tag to fluoresce • Fluorescence increases with incorporation into DNA product
  • 78. and can be quantified 4-41 4.3 Methods of Expressing Cloned Genes Cloning a gene permits • Production of large quantities of a particular DNA sequence for detailed study • Large quantities of the gene’s product can also be obtained for further use – Study – Commerce 4-42 Expression Vectors • Vectors discussed so far are used to first put a foreign DNA into a bacterium to replicate and screen • Expression vectors are those that can yield protein products of the cloned genes
  • 79. – Bacterial expression vectors typically have two elements required for active gene expression; a strong promoter and a ribosome binding site near an initiating codon 4-43 Controlling the lac Promoter • lac promoter is somewhat inducible – Stays off until stimulated by inducer IPTG – However, repression is typically incomplete or leaky and some expression will still occur • To avoid this problem, use a plasmid or phagemid carrying its own lacI repressor gene to keep the cloned gene off until it is induced by IPTG 4-44 Alternative to the lac Promoter • Promoter from ara operon, PBAD, allow fine control of transcription – Inducible by arabinose, a sugar – Transcription rate varies with arabinose
  • 80. concentration 4-45 Summary • Expression vectors are designed to yield the protein product of a cloned gene • To optimize expression, these vectors include strong bacterial or phage promoters and bacterial ribosome binding sites • Most cloning vectors are inducible, which avoids premature overproduction of a foreign product that could poison the bacterial host cells 4-46 Expression Vectors That Produce Fusion Proteins • Most vectors express fusion proteins – The actual natural product of the gene isn’t made – Extra amino acids help in purifying the protein product
  • 81. • Oligohistidine expression vector has a short sequence just upstream of MCS encoding 6 His – Oligohistidine has a high affinity for divalent metal ions like nickel (Ni2+) – Permits purification by nickel affinity chromatography – The his tag can be removed using enzyme enterokinase without damage to the protein product 4-47 Using an Oligohistidine Expression Vector 4-48 Expression vector lgt11 • This phage contains the lac control region followed by the lacZ gene • The cloning sites are located within the lacZ gene
  • 82. • Products of gene correctly inserted will be fusion proteins with a b-galactosidase leader 4-49 Detecting positive lgt11 clones via antibody screening • Lambda phages with cDNA inserts are plated • Protein released are blotted onto a support • Probe with antibody specific to protein • Antibody bound to protein from plaque is detected with labeled protein A 4-50
  • 83. Summary • Expression vectors frequently produce fusion proteins with one part of the protein coming from the coding sequences in the vector and the other part from sequences in the cloned gene • Many fusion proteins have advantage of being simple to isolate by affinity chromatography • Vector lgt11 produces fusion proteins that can be detected in plaques with a specific antiserum 4-51 Bacterial Expression System Shortcomings • There are problems with expression of eukaryotic proteins in a bacterial system – Bacteria may recognize the proteins as foreign and destroy them – Post-translational modifications are different in bacteria – Bacterial environment may not permit correct protein folding • Very high levels of cloned eukaryotic proteins can be expressed in useless,
  • 84. insoluble form 4-52 Eukaryotic Expression Systems • Avoid bacterial expression problems by expressing the protein in a eukaryotic cell • Initial cloning done in E. coli using a shuttle vector, able to replicate in both bacterial and eukaryotic cells • Yeast is suited for this purpose – Rapid growth and ease of culture – A eukaryote with more appropriate post- translational modification – Use of the yeast export signal peptide secretes protein into growth medium for easy purification 4-53 Use of Baculovirus As Expression Vector • Viruses in this class have a large circular DNA genome, 130 kb • Major viral structural protein is made in huge amounts in infected cells – The promoter for this protein, polyhedrin, is
  • 85. very active – These vectors can produce up to 0.5 g of protein per liter of medium – Nonrecombinant viral DNA entering cells does not result in infectious virus as it lacks an essential gene supplied by the vector 4-54 Animal Cell Transfection • Carried out in two ways: • Calcium phosphate – Mix cells with DNA in a phosphate buffer and add a solution of calcium salt to form a precipitate – The cells take up the calcium phosphate crystals, which include some DNA • Liposomes – The DNA is mixed with lipid to form liposomes, small vesicles with some of the DNA inside – DNA-bearing liposomes fuse with the cell membrane to deliver DNA inside the cell 4-55
  • 86. Summary • Foreign genes can be expressed in eukaryotic cells • These eukaryotic systems have advantages over prokaryotic systems for producing eukaryotic proteins – The proteins tend to fold properly and are soluble, rather than aggregated into insoluble inclusion bodies – Post-translational modifications are compatible 4-56 Using the Ti Plasmid to Transfer Genes to Plants • Genes can be introduced into plants with vectors that can replicate in plant cells • Common bacterial vector promoters and replication origins are not recognized by plant cells • Plasmids are used containing T-DNA – T-DNA is derived from a plasmid known as tumor-inducing (Ti)
  • 87. – Ti plasmid comes from bacteria that cause plant tumors called crown galls 4-57 Ti Plasmid Infection • Bacterium infects plant, transfers Ti plasmid to host cells • T-DNA integrates into the plant DNA causing abnormal proliferation of plant cells • T-DNA genes direct the synthesis of unusual organic acids, opines which can serve as an energy source to the infecting bacteria but are useless to the plant 4-58 The Ti Plasmid Transfers Crown Gall 4-59 Use of the T-DNA Plasmid 4-60
  • 88. Summary • Molecular biologists can transfer cloned genes to plants, creating transgenic organisms with altered characteristics, using a plant vector such as the Ti plasmid Molecular Biology Fifth Edition Chapter 5 Molecular Tools for Studying Genes and Gene Activity Lecture PowerPoint to accompany Robert F. Weaver Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5-2 5.1 Molecular Separations • Often mixtures of proteins or nucleic acids are generated during the course of
  • 89. molecular biological procedures – A protein may need to be purified from a crude cellular extract – A particular nucleic acid molecule made in a reaction needs to be purified • Gel electrophoresis is used to separate different species of: – Nucleic acid – Protein 5-3 DNA Gel Electrophoresis • Melted agarose is poured into a form equipped with removable comb • Comb “teeth” form slots in the solidified agarose • DNA samples are placed in the slots • An electric current is run through the gel at a neutral pH to allow the sample to
  • 90. travel through the gel matrix 5-4 DNA Separation by Agarose Gel Electrophoresis • DNA is negatively charged due to the phosphates in its backbone and moves toward the positive pole – Small DNA pieces have little frictional drag so they move rapidly – Large DNAs have more frictional drag so their mobility is slower – Distributes DNA according to size • Largest near the top • Smallest near the bottom • DNA is stained with fluorescent dye that intercalates between the bases 5-5 DNA Size Estimation
  • 91. • Mobility of fragments are plotted v. log of molecular weight (or number of base pairs) • Electrophoresis of unknown DNA in parallel with standard fragments permits size estimation upon comparison • Same principles apply to RNA separation 5-6 Electrophoresis of Large DNA • Special techniques are required for DNA fragments larger than about 1 kilobases • Instead of constant current, alternate long pulses of current in forward direction with shorter pulses in either opposite or sideways direction • Technique is called pulsed-field gel electrophoresis (PFGE)
  • 92. 5-7 Protein Gel Electrophoresis • Separation of proteins is done using polyacrylamide gel electrophoresis (PAGE) – Treat proteins to denature subunits with detergent such as sodium dodecyl sulfate (SDS) • SDS coats polypeptides with negative charges so all move to anode • Masks natural charges of protein subunits so all move relative to mass not charge – As with DNA smaller proteins move faster toward the anode 5-8 Summary • DNAs, RNAs, and proteins of various masses can be separated by gel electrophoresis • Most common gel used in nucleic acid electrophoresis is agarose but polyacrylamide is typically used in protein electrophoresis
  • 93. • SDS-PAGE is used to separate polypeptides according to their masses 5-9 Two-Dimensional Gel Electrophoresis • While SDS-PAGE gives good resolution of polypeptides, some mixtures are so complex that additional resolution is needed • Two-dimensional gel electrophoresis: – Nondenaturing gel electrophoresis (no SDS) uses 2 consecutive gels each in a different dimension – Sequential gels with distinct pH separation and polyacrylamide gel concentration 5-10 Two-Dimensional Gel Electrophoresis Technique A two process method: • Isoelectric focusing gel: mixture of proteins electrophoresed through gel in a narrow
  • 94. tube containing a pH gradient – Negatively charged protein moves to its isoelectric point at which it is no longer charged – Tube gel is removed and used as the sample in the second process 5-11 • Standard SDS-PAGE: – Tube gel is removed and used as the sample at the top of a standard polyacrylamide gel – Proteins partially resolved by isoelectric focusing are further resolved according to size • When used to a compare complex mixtures of proteins prepared under two different conditions, even subtle differences are visible Two-Dimensional Gel Electrophoresis Technique continued 5-12 Ion-Exchange Chromatography
  • 95. • Chromatography originally referred to the pattern seen after separating colored substances on paper • Ion-exchange chromatography uses a resin to separate substances by charge • This is especially useful for proteins • Resin is placed in a column and the sample is loaded onto the column material 5-13 Separation by Ion-Exchange Chromatography • Once the sample is loaded buffer is passed over the resin + sample • As ionic strength of elution buffer increases, samples of solution flowing through the column are collected • Samples are tested for the presence of the protein of interest
  • 96. 5-14 Gel Filtration Chromatography • Protein size is a valuable property that can be used as a basis of physical separation • Gel filtration uses columns filled with porous resins that let in smaller substances and exclude larger substances • As a result larger substances travel faster through the column 5-15 Affinity Chromatography • The resin contains a substance to which the molecule of interest has a strong and specific affinity • The molecule binds to a column resin coupled to the affinity reagent – Molecule of interest is retained – Most other molecules flow through without binding – Last, the molecule of interest is eluted from the column using a specific solution that disrupts their specific binding
  • 97. 5-16 Summary • High-resolution separation of proteins can be achieved by two-dimensional gel electrophoresis • Ion-exchange chromatography can be used to separate substances according to their sizes • Gel filtration chromatography uses columns filled with porous resins that let in smaller substances but exclude larger ones • Affinity chromatography is a powerful purification technique that exploits an affinity reagent with strong and specific affinity for a molecule of interest 5-17 5.2 Labeled Tracers • For many years “labeled” has been synonymous with “radioactive” • Radioactive tracers allow vanishingly small quantities of substances to be detected
  • 98. • Molecular biology experiments typically require detection of extremely small amounts of a particular substance 5-18 Autoradiography Autoradiography is a means of detecting radioactive compounds with a photographic emulsion – Preferred emulsion is x-ray film – DNA is separated on a gel and radiolabeled – Gel is placed in contact with x- ray film for hours or days – Radioactive emissions from the labeled DNA expose the film – Developed film shows dark bands 5-19 Autoradiography Analysis
  • 99. • Relative quantity of radioactivity can be assessed looking at the developed film • More precise measurements are made using a densitometer – Area under peaks on a tracing by a scanner – Proportional to darkness of the bands on autoradiogram 5-20 Liquid Scintillation Counting Radioactive emissions from a sample create photons of visible light are detected by a photomultiplier tube in the process of liquid scintillation counting – Remove the radioactive material (band from gel) to a vial containing scintillation fluid – Fluid contains a fluor that fluoresces when hit with radioactive emissions – Acts to convert invisible radioactivity into visible light
  • 100. 5-21 Nonradioactive Tracers • Newer nonradioactive tracers now rival older radioactive tracers in sensitivity • These tracers do not have hazards: – Health exposure – Handling – Disposal • Increased sensitivity is from use of a multiplier effect of an enzyme that is coupled to probe for molecule of interest 5-22 Detecting Nucleic Acids With a Nonradioactive Probe 5-23 5.3 Using Nucleic Acid Hybridization • Hybridization is the ability of one single- stranded nucleic acid to form a double helix with another single strand of
  • 101. complementary base sequence • Previous discussion focused on colony and plaque hybridization • This section looks at techniques for isolated nucleic acids 5-24 Southern Blots: Identifying Specific DNA Fragments • Digests of genomic DNA are separated on a gel • The separated pieces are transferred to filter (nitrocellulose) by diffusion, or more recently by electrophoresing the DNA onto the filter • The filter is then treated with alkali to denature the DNA, resulting ssDNA binds to the filter • A labeled cDNA probe that is complementary to the DNA of interest is then applied to the filter • A positive band should be detectable where hybridization between the probe and DNA occurred 5-25
  • 102. Southern Blots • The probe hybridizes and a band is generated corresponding to the DNA fragment of interest • Visualize bands with x-ray film or autoradiography • Multiple bands can lead to several interpretations – Multiple genes – Several restriction sites in the gene 5-26 DNA Fingerprinting and DNA Typing • Southern blots are used in forensic labs to identify individuals from DNA-containing materials • Minisatellite DNA is a sequence of bases repeated several times, also called a DNA fingerprint – Individuals differ in the pattern of repeats of the basic sequence
  • 103. – The difference is large enough that 2 people have only a remote chance of having exactly the same pattern of repeats 5-27 DNA Fingerprinting Process is a Southern blot • Cut the DNA under study with restriction enzyme – Ideally cut on either side of minisatellite but not inside • Run the digested DNA on a gel and blot • Probe with labeled minisatellite DNA and image – Note that real samples result in very complex patterns 5-28 Forensic Uses of DNA Fingerprinting
  • 104. and DNA Typing • While people have different DNA fingerprints, parts of the pattern are inherited in a Mendelian fashion – Can be used to establish parentage – Potential to identify criminals – Remove innocent people from suspicion • Actual pattern has so many bands they can smear together indistinguishably – Forensics uses probes for just a single locus – Set of probes gives a set of simple patterns 5-29 In Situ Hybridization: Locating Genes in Chromosomes • Labeled probes can be used to hybridize to chromosomes and reveal which chromosome contains the gene of interest – Spread chromosomes from a cell – Partially denature DNA creating single-stranded regions to hybridize to labeled probe – Stain chromosomes and detect presence of label on particular chromosome • Probe can be detected with a fluorescent
  • 105. antibody in a technique called fluorescence in situ hybridization (FISH) 5-30 Immunoblots Immunoblots (also called Western blots) use a similar process to Southern blots – Electrophoresis of proteins – Blot the proteins from the gel to a membrane – Detect the protein using antibody or antiserum to the target protein – Labeled secondary antibody is used to bind the first antibody for visualization and to increase the signal 5-31 Summary • Labeled DNA (or RNA) probes can be used to hybridize to DNAs of the same or very similar sequence on a Southern blot • DNA fingerprinting can be used as a forensic tool or to test parentage • In situ hybridization can be used to locate genes
  • 106. or other specific DNA sequences on whole chromosomes • Proteins can be detected and quantified in a complex mixture using Western blots 5-32 5.4 DNA Sequencing • Sanger, Maxam, and Gilbert developed 2 methods for determining the exact base sequence of a cloned piece of DNA • Modern DNA sequencing is based on the Sanger method and uses dideoxy nucleotides to terminate DNA synthesis – The process yields a series of DNA fragments whose size is measured by electrophoresis – The last base in each fragment is known as that dideoxy nucleotide was used to terminate the reaction – Ordering the fragments by size tells the base sequence of the DNA
  • 107. 5-33 Sanger Method of DNA Sequencing 5-34 Automated DNA Sequencing • Manual sequencing is powerful but slow • Automated sequencing uses dideoxynucleotides tagged with different fluorescent molecules – Products of each dideoxynucleotide will fluoresce a different color – Four reactions are completed, then mixed together and run out on one lane of a gel 5-35 High Throughput Sequencing • Once an organism’s genome sequence is known, very rapid sequencing techniques can be applied
  • 108. to sequence the genome of another member of the same species • Produces relatively short reads or contiguous sequences (25-35bp or 200-300bp, depending on the method) that can easily be pieced together if a reference sequence is available 5-36 High Throughput Sequencing • Pyrosequencing is one example that is an automated system with the advantages of speed and accuracy - nucleotides are added one by one and the incorporation of a nucleotide is detected by a release of pyrophosphate, which leads to a flash of light • Another method (Illumina company) starts by attaching short pieces of DNA to a solid surface, amplifying each DNA in a tiny patch on the surface, then sequencing the patches together by extending them one nucleotide at a time using fluorescent chain-terminating nucleotides, whose fluoresce reveals their identity
  • 109. 5-37 Restriction Mapping • Prior to the start of large-scale sequencing preliminary work is done to locate landmarks – A map based on physical characteristics is called a physical map – If restriction sites are the only map features then a restriction map has been prepared 5-38 Restriction Map Example • Consider a 1.6 kb piece of DNA as an example • Cut separate samples of the original 1.6 kb fragment with different restriction enzymes • Separate the digests on an agarose gel to determine the size of pieces from each digest • Can also use same digest to
  • 110. find the orientation of an insert cloned into a vector 5-39 Southern Blots and Restriction Mapping 5-40 Summary • Physical maps tell about the spatial arrangement of physical “landmarks” such as restriction sites – In restriction mapping cut the DNA in question with 2 or more restriction enzymes in separate reactions – Measure the sizes of the resulting fragments – Cut each with another restriction enzyme and measure size of subfragments by gel electrophoresis • Sizes permit location of some restriction sites relative to others • Improve process by Southern blotting fragments and hybridizing them to labeled fragments from another restriction enzyme to reveal overlaps
  • 111. 5-41 5.5 Protein Engineering With Cloned Genes: Site-Directed Mutagenesis • Cloned genes permit biochemical microsurgery on proteins – Specific bases in a gene may be changed – Amino acids at specific sites in the protein product may be altered as a result – Effects of those changes on protein function can be observed 5-42 PCR-based Site-Directed Mutagenesis 5-43 Summary • Using cloned genes, one can introduce changes that may alter the amino acid sequence of the corresponding protein products • Mutagenized DNA can be made with: – Double-stranded DNA – Two complementary mutagenic primers
  • 112. – PCR • Digest the PCR product to remove wild-type DNA • Cells can be transformed with mutagenized DNA 5-44 5.6 Mapping and Quantifying Transcripts • In the field of molecular biology mapping (locating start and end) and quantifying (how much transcript exists at a set time) transcripts are common procedures • Often transcripts do not have a uniform terminator, resulting in a continuum of species smeared on a gel • Techniques that are specific for the sequence of interest are important 5-45 Northern Blots • Northern blots detect RNA • Example: You have cloned a cDNA – Question: How actively is the corresponding gene expressed in different tissues?
  • 113. – Answer: Find out using a Northern Blot • Obtain RNA from different tissues • Run RNA on agarose gel and blot to membrane • Hybridize to a labeled cDNA probe – Northern plot tells abundance of the transcript – Quantify using densitometer 5-46 S1 Mapping Use S1 mapping to locate the ends of RNAs and to determine the amount of a given RNA in cells at a given time – Label a ssDNA probe that can only hybridize to transcript of interest – Probe must span the sequence start to finish – After hybridization, treat with S1 nuclease which degrades ssDNA and RNA – Transcript protects part of the probe from degradation – Size of protected area can be measured by gel electrophoresis 5-47
  • 114. Summary • A Northern blot is similar to a Southern blot but is a method used for detection of RNA • In S1 mapping, a labeled DNA probe is used to detect 5’- or 3’-end of a transcript • Amount of probe protected is proportional to concentration of transcript, so S1 mapping can be quantitative • RNase mapping is a variation on SI mapping that uses an RNA probe and RNase 5-48 Run-Off Transcription • A good assay to measure the rate of in vitro transcription • DNA fragment containing gene to transcribe is cut with restriction enzyme in middle of transcription region • Transcribe the truncated fragment in vitro using labeled nucleotides, as polymerase
  • 115. reaches truncation it “runs off” the end • Measure length of run-off transcript compared to location of restriction site at 3’- end of truncated gene 5-49 Summary • Run-off transcription is a means of checking efficiency and accuracy of in vitro transcription – Gene is truncated in the middle and transcribed in vitro in presence of labeled nucleotides – RNA polymerase runs off the end making an incomplete transcript – Size of run-off transcript locates transcription start site – Amount of transcript reflects efficiency of transcription 5-50 5.7 Measuring Transcription Rates in Vivo • Primer extension, S1 mapping and Northern blotting will determine the concentration of specific transcripts at a
  • 116. given time • These techniques do not really reveal the rate of transcript synthesis as concentration involves both: – Transcript synthesis – Transcript degradation 5-51 Reporter Gene Transcription • Place a surrogate reporter gene under the control of a specific promoter and measure the accumulation of the product of this reporter gene • The reporter genes are carefully chosen to have products very convenient to assay – lacZ produces b-galactosidase which has a blue cleavage product – cat produces chloramphenicol acetyl transferase (CAT) which inhibits bacterial growth – Luciferase produces a chemiluminescent compound that emits light 5-52
  • 117. Measuring Protein Accumulation in Vivo • Gene activity can be monitored by measuring the accumulation of protein, the ultimate gene product • There are two primary methods of measuring protein accumulation – Immunoblotting / Western blotting (discussed earlier) – Immunoprecipitation • Immunoprecipitation typically uses an antibody that will bind specifically to the protein of interest followed with a secondary antibody complexed to Protein A on resin beads using a low-speed centrifuge 5-53 5.8 Assaying DNA-Protein Interactions • Study of DNA-protein interactions is of significant interest to molecular biologists • Types of interactions often studied: – Protein-DNA binding – Which bases of DNA interact with a protein 5-54
  • 118. Filter Binding Filter binding is used to measure DNA- protein interaction and based on the fact that double-stranded DNA will not bind by itself to a filter, but a protein-DNA complex will – Double-stranded DNA can be labeled and mixed with protein – Assay protein-DNA binding by measuring the amount of label retained on the filter 5-55 Nitrocellulose Filter-Binding Assay • dsDNA is labeled and mixed with protein • Pour dsDNA through a nitrocellulose filter • Measure amount of radioactivity that passed through filter and retained on filter 5-56 Gel Mobility Shift • DNA moves through a gel faster when it is not
  • 119. bound to protein • Gel shift assays detect interaction between protein and DNA by reduction of the electrophoretic mobility of a small DNA bound to a protein 5-57 Footprinting • Footprinting detects protein-DNA interaction and will show where a target lies on DNA and which bases are involved in protein binding • Three methods are very popular: – DNase footprinting – Dimethylsulfate footprinting – Hydroxyl radical footprinting 5-58 DNase Footprinting Protein binding to DNA covers the binding site and protects from attack by DNase
  • 120. • End label DNA, 1 strand only • Protein binds DNA • Treat complex with DNase I mild conditions for average of 1 cut per molecule • Remove protein from DNA, separate strands and run on a high-resolution polyacrylamide gel 5-59 Summary • Footprinting finds target DNA sequence or binding site of a DNA-binding protein • DNase footprinting binds protein to end-labeled DNA target, then attacks DNA-protein complex with DNase • DNA fragments are electrophoresed with protein binding site appearing as a gap in the pattern where protein protected DNA from degradation
  • 121. 5-60 Chromatin Immunoprecipitation (ChIP) • ChIP is a method used to discover whether a given protein is bound to a given gene in chromatin - the DNA-protein complex that is the natural state of the DNA in a living cell • ChIP uses an antibody to precipitate a particular protein in complex with DNA, and PCR to determine whether the protein binds near a particular gene 5-61 Chromatin Immunoprecipitation (ChIP) 5-62 5.9 Assaying Protein-Protein Interactions • Immunoprecipitation uses an antibody that will bind specifically to the protein of interest and, using a low-speed centrifuge, will ‘pull-down’ any proteins associated with the protein of interest
  • 122. 5-63 5.11 Knockouts and Transgenes • Probing structures and activities of genes does not answer questions about the role of the gene in the life of the organism • Targeted disruption of genes is now possible in several organisms • When genes are disrupted in mice the products are called knockout mice • Foreign genes, called transgenes, can also be added to an organism, such as a mouse, to create transgenic mice 5-64 Knockout Results • Phenotype may not be obvious in the progeny, but still instructive • Other cases can be lethal with the mice dying before birth • Intermediate effects are also common and may require monitoring during the life of
  • 123. the mouse 5-65 Methods to Generate Transgenic Mice • Two methods to generate transgenic mice: • 1. Injection of cloned foreign gene into the sperm pronucleus just after fertilization of a mouse egg but before the sperm and egg nuclei have fused to allow for insertion of the foreign DNA into the embryonic cell DNA • 2. Injection of cloned foreign DNA into mouse embryonic stem cells, creating transgenic ES cells • Both methods produce chimeric mice that must undergo several rounds of breeding and selection to find true transgenic animals 5-66 Summary • To probe the role of a gene, molecular
  • 124. biologists can perform targeted disruption of the corresponding gene in a mouse and then look for the effects of the mutation in the ‘knockout mouse’ or insert the foreign gene as a transgene in the ‘transgenic mouse’ Molecular Biology Fifth Edition Chapter 6 The Mechanism of Transcription in Bacteria Lecture PowerPoint to accompany Robert F. Weaver Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 6-2 6.1 RNA Polymerase Structure By 1969 SDS-PAGE of RNA polymerase from E. coli had shown several subunits
  • 125. – 2 very large subunits are b (150 kD) and b’ (160 kD) – Sigma (s) at 70 kD – Alpha (a) at 40 kD – 2 copies present in holoenzyme – Omega (ω) at 10 kD • Was not clearly visible in SDS-PAGE, but seen in other experiments • Not required for cell viability or in vivo enzyme activity • Appears to play a role in enzyme assembly 6-3 Sigma as a Specificity Factor • Core enzyme without the s subunit could not transcribe viral DNA, yet had no problems with highly nicked calf thymus DNA • With s subunit, the holoenzyme worked equally well on both types of DNA 6-4
  • 126. Summary • The key player in the transcription process is RNA polymerase • The E. coli enzyme is composed of a core, which contains the basic transcription machinery, and a s-factor, which directs the core to transcribe specific genes 6-5 6.2 Promoters • Why was the core RNA polymerase capable of transcribing nicked DNA in the previous table? • Nicks and gaps are good sites for RNA polymerase to bind nonspecifically • The presence of the s-subunit permits recognition of authentic RNA polymerase binding sites called promoters • Transcription that begins at promoters is specific, directed by the s-subunit 6-6 Binding of RNA Polymerase to Promoters
  • 127. • How tightly does core enzyme v. holoenzyme bind DNA? • Experiment measures binding of DNA to enzyme using nitrocellulose filters – Holoenzyme binds filters tightly – Core enzyme binding is more transient 6-7 Temperature and RNA Polymerase Binding • As the temperature is lowered, the binding of RNA polymerase to DNA decreases dramatically • Higher temperatures promote DNA melting and encourage RNA
  • 128. polymerase binding 6-8 RNA Polymerase Binding Hinkle and Chamberlin proposed: • RNA polymerase holoenzyme binds DNA loosely at first – Binds at promoter initially – Scans along the DNA until it finds a promoter • Complex with holoenzyme loosely bound at the promoter is a closed promoter complex as DNA is in a closed ds form • Holoenzyme can then melt a short DNA region at the promoter to form an open promoter complex with polymerase bound tightly to DNA 6-9 Polymerase/Promoter Binding • Holoenzyme binds DNA loosely at first
  • 129. • Complex loosely bound at promoter = closed promoter complex, dsDNA in closed form • Holoenzyme melts DNA at promoter forming open promoter complex - polymerase tightly bound 6-10 Summary • The s-factor allows initiation of transcription by causing the RNA polymerase holoenzyme to bind tightly to a promoter • This tight binding depends on local melting of the DNA to form an open promoter complex and is stimulated by s • The s-factor can therefore select which genes will be transcribed 6-11
  • 130. Core Promoter Elements • There is a region common to bacterial promoters described as 6-7 bp centered about 10 bp upstream of the start of transcription = -10 box • Another short sequence centered 35 bp upstream is known as the -35 box • Comparison of thousands of promoters has produced a consensus sequence (or most common sequence) for each of these boxes 6-12 Promoter Strength • Consensus sequences: – -10 box sequence approximates TATAAT – -35 box sequence approximates TTGACA • Mutations that weaken promoter binding: – Down mutations – Increase deviation from the consensus sequence • Mutations that strengthen promoter binding: – Up mutations – Decrease deviation from the consensus sequence
  • 131. 6-13 UP Element • The UP element is upstream of the core promoter, stimulating transcription by a factor of 30 • UP is associated with 3 “Fis” sites which are binding sites for the transcription- activator protein Fis, not for the polymerase itself 6-14 The rrnB P1 Promoter • Transcription from the rrn promoters respond –Positively to increased concentration of iNTP –Negatively to the alarmone ppGpp 6-15 6.3 Transcription Initiation • Transcription initiation was assumed to end as RNA polymerase formed 1st phosphodiester bond
  • 132. • Carpousis and Gralla found that very small oligonucleotides (2-6 nt long) are made without RNA polymerase leaving the DNA • Abortive transcripts such as these have been found up to 10 nt 6-16 Stages of Transcription Initiation • Formation of a closed promoter complex • Conversion of the closed promoter complex to an open promoter complex • Polymerizing the early nucleotides – polymerase at the promoter • Promoter clearance – transcript becomes long enough to form a stable hybrid with template 6-17 Reuse of s
  • 133. • During initiation s can be recycled for additional use with a new core polymerase • The core enzyme can release s which is then free to associate with another core enzyme 6-18 Models for the s-Cycle • The obligate release version of the s-cycle model arose from experiments performed by Travers and Burgess that proposed the dissociation of s from core as polymerase undergoes promoter clearance and switches from initiation to elongation mode • The stochastic release model proposes that s is indeed released from the core polymerase but that there is no discrete point of release during transcription and that the release occurs at random - a preponderance of evidence favors this model
  • 134. 6-19 Local DNA Melting at the Promoter • From the number of RNA polymerase holoenzymes bound to DNA, it was calculated that each polymerase caused a separation of about 10 bp • In another experiment, the length of the melted region was found to be 12 bp • Later, size of the DNA transcription bubble in complexes where transcription was active was found to be 17-18 bp 6-20 Promoter Clearance • RNA polymerases have evolved to recognize and bind strongly to promoters • This poses a challenge when it comes time for promoter clearance as those strong bonds must be broken in order for polymerase to leave the promoter and enter the elongation phase 6-21 Promoter Clearance
  • 135. • Several hypotheses have been proposed • The polymerase cannot move enough downstream to make a 10-nt transcript without doing one of three things: - transient excursion: moving briefly downstream and then snapping back to the starting position - inchworming: stretching itself by leaving its trailing edge in place while moving its leading edge downstream - scrunching: compressing the DNA without moving itself 6-22 Abortive Transcription, Scrunching and Promoter Clearance • Ebert and colleagues performed several experiments to distinguish between the hypotheses • Using E.coli polymerase the authors concluded that approximately 100% of all transcription cycles involved scrunching, which suggested that scrunching is required for promoter clearance • The E.coli polymerase achieves abortive transcription by scrunching: drawing downstream
  • 136. DNA into the polymerase without actually moving and losing its grip on promoter DNA • The scrunched DNA could store enough energy to allow the polymerase to break its bonds to the promoter and begin productive transcription 6-23 Structure and Function of s • Genes encoding a variety of s-factors have been cloned and sequenced • There are striking similarities in amino acid sequence clustered in 4 regions • Conservation of sequence in these regions suggests important function • All of the 4 sequences are involved in binding to core and DNA 6-24 Homologous Regions in Bacterial s Factors 6-25 E. coli s70
  • 137. • Four regions of high sequence similarity are indicated • Specific areas that recognize the core promoter elements are the -10 box and – 35 box 6-26 Region 1 • Role of region 1 appears to be in preventing s from binding to DNA by itself • This is important as s binding to promoters could inhibit holoenzyme binding and thereby inhibit transcription Region 2 • This region is the most highly conserved of the four • There are four subregions – 2.1 to 2.4 • 2.4 recognizes the promoter’s -10 box • The 2.4 region appears to be a-helix 6-27 Regions 3 and 4 • Region 3 is involved in both core and
  • 138. DNA binding • Region 4 is divided into 2 subregions – This region seems to have a key role in promoter recognition – Subregion 4.2 contains a helix-turn-helix DNA-binding domain and appears to govern binding to the -35 box of the promoter 6-28 Summary • Comparison of different s gene sequences reveals 4 regions of similarity among a wide variety of sources • Subregions 2.4 and 4.2 are involved in promoter -10 box and -35 box recognition • The s-factor by itself cannot bind to DNA, but DNA interaction with core unmasks a DNA- binding region of s • Region between amino acids 262 and 309 of b’ stimulates s binding to the nontemplate strand in the -10 region of the promoter
  • 139. 6-29 Role of a-Subunit in UP Element Recognition • RNA polymerase itself can recognize an upstream promoter element, UP element • While s-factor recognizes the core promoter elements, what recognizes the UP element? • It appears to be the a-subunit of the core polymerase 6-30 Modeling the Function of the C- Terminal Domain • RNA polymerase binds to a core promoter via its s- factor, no help from C- terminal domain of a-subunit • Binds to a promoter with an UP element using s plus the a-subunit C-terminal domains (CTD) • Results in very strong
  • 140. interaction between polymerase and promoter • This produces a high level of transcription 6-31 6.4 Elongation • After transcription initiation is accomplished, core polymerase continues to elongate the RNA • Nucleotides are added sequentially, one after another in the process of elongation 6-32 Function of the Core Polymerase • Core polymerase contains the RNA synthesizing machinery • Phosphodiester bond formation involves the b- and b’-subunits • These subunits also participate in DNA binding • Assembly of the core polymerase is a
  • 141. major role of the a-subunit 6-33 Role of b in Phosphodiester Bond Formation • Core subunit b lies near the active site of the RNA polymerase • This active site is where the phosphodiester bonds are formed linking the nucleotides • The s-factor may also be near the nucleotide-binding site during the initiation phase 6-34 Structure of the Elongation Complex • This section will examine how well predictions have been borne out by structural studies • How does the polymerase deal with problems of unwinding and rewinding templates? • How does it move along the helical template without twisting RNA product
  • 142. around the template? 6-35 RNA-DNA Hybrid • The area of RNA-DNA hybridization within the E. coli elongation complex extends from position –1 to –8 or –9 relative to the 3’ end of the nascent RNA • In T7 the similar hybrid appears to be 8 bp long 6-36 Structure of the Core Polymerase • X-ray crystallography on the Thermus aquaticus RNA polymerase core reveals an enzyme shaped like a crab claw • It appears designed to grasp the DNA • A channel through the enzyme includes the catalytic center – Mg2+ ion coordinated by 3 Asp residues – Rifampicin-binding site
  • 143. 6-37 Structure of the Holoenzyme-DNA Complex Crystal structure of T. aquaticus holoenzyme-DNA complex as an open promoter complex reveals: – DNA is bound mainly to s-subunit – Interactions between amino acids in region 2.4 of s and -10 box of promoter are possible – 3 highly conserved aromatic amino acids are able to participate in promoter melting as predicted – 2 invariant basic amino acids in s predicted to function in DNA binding are positioned to do so – A form of the polymerase that has 2 Mg2+ ions 6-38 Structure of the Elongation Complex • The X-ray crystal structure of the Thermus thermophilus RNA polymerase elongation complex in 2007 revealed several important observations – a valine residue in the E’ subunit inserts into the minor groove of the downstream DNA – the downstream DNA is double-stranded up
  • 144. to and including the +2 base pair – the enzyme can accommodate nine base pairs of RNA-DNA hybrid – the RNA product in the exit channel is twisted into the shape it would assume as 1/2 of an A-form dsRNA 6-39 Topology of Elongation • Elongation of transcription involves polymerization of nucleotides as the RNA polymerase travels along the template DNA • Polymerase maintains a short melted region of template DNA • DNA must unwind ahead of the advancing polymerase and rewind behind it • Strain introduced into the template DNA ahead of the transcription bubble is relaxed by topoisomerases 6-40 Pausing and Proofreading
  • 145. • RNA polymerase frequently pauses, or even backtracks, during elongation • Pausing allows ribosomes to keep pace with the RNA polymerase, and it is the first step in termination • Backtracking aids proofreading by extruding the 3’-end of the RNA out of the polymerase, where misincorporated nucleotides can be removed by an inherent nuclease activity of the polymerase, stimulated by auxiliary factors 6-41 6.5 Termination of Transcription • When the polymerase reaches a terminator at the end of a gene it falls off the template and releases the RNA • There are 2 main types of terminators – Intrinsic terminators function with the RNA polymerase by itself without help from other proteins – Other type depends on auxiliary factor called -dependent
  • 146. terminators 6-42 Rho-Independent Termination • Intrinsic or rho-independent termination depends on terminators of 2 elements: – Inverted repeats followed immediately by – T-rich region in the nontemplate strand of the gene • An inverted repeat predisposes a transcript to form a hairpin structure due to complementary base pairing between the inverted repeat sequences 6-43 Inverted Repeats and Hairpins • The repeat at right is symmetrical around its center shown with a dot • A transcript of this sequence is self-
  • 147. complementary – Bases can pair up to form a hairpin as seen in the lower panel 6-44 Model of Intrinsic Termination Bacterial terminators act by: • Base-pairing of something to the transcript to destabilize RNA-DNA hybrid – Causes hairpin to form • This causes transcription to pause – a string of U’s incorporated just downstream of hairpin to destabilize the hybrid and the RNA falls off the DNA template 6-45
  • 148. Rho-Dependent Termination • Rho caused depression of the ability of RNA polymerase to transcribe phage DNAs in vitro • This depression was due to termination of transcription • After termination, polymerase must reinitiate to begin transcribing again 6-46 Rho Affects Chain Elongation • There is little effect of rho or r on transcription initiation, if anything it is increased • The effect of rho or r on total RNA synthesis is a significant decrease • This is consistent with action of rho or r to terminate transcription forcing time- consuming reinitiation 6-47 Rho Causes Production of Shorter Transcripts
  • 149. • Synthesis of much smaller RNAs occurs in the presence of rho or r compared to those made in the absence • To ensure that this due to r itself and not to RNase activity of r, RNA was transcribed without r and then incubated in the presence of r • There was no loss of transcript size, so no RNase activity in r 6-48 Rho Releases Transcripts from the DNA Template • Compare the sedimentation of transcripts made in presence and absence of r – Without r, transcripts cosedimented with the DNA template – they hadn’t been released – With r present in the incubation, transcripts sedimented more slowly – they were not associated with the DNA template • It appears that r serves to release the RNA transcripts from the DNA template 6-49
  • 150. Mechanism of Rho • No string of T’s in the r- dependent terminator, just inverted repeat to hairpin • Binding to the growing transcript, r follows the RNA polymerase • It catches the polymerase as it pauses at the hairpin • Releases transcript from the DNA-polymerase complex by unwinding the RNA-DNA hybrid 6-50 Summary • Using the trp attenuator as a model rho-independet terminator revealed two important features: 1 - an inverted repeat that allows a hairpin to for at the end of the transcript 2 - a string of T’s in the nontemplate strand that results in a string of weak rU-dA base pairs holding the transcript to the template strand
  • 151. • Rho-dependent terminators consist of an inverted repeat, which can cause a hairpin to form in the transcript but no string of T’s Molecular Biology Fifth Edition Chapter 7 Operons: Fine Control of Bacterial Transcription Lecture PowerPoint to accompany Robert F. Weaver Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 7-2 7.1 The lac Operon • The lac operon was the first operon
  • 152. discovered • It contains 3 genes coding for E. coli proteins that permit the bacteria to use the sugar lactose – Galactoside permease (lacY) which transports lactose into the cells -galactosidase (lacZ) cuts the lactose into galactose and glucose – Galactoside transacetylase (lacA) whose function is unclear 7-3 Genes of the lac Operon • The genes are grouped together: – lacZ = b-galactosidase – lacY = galactoside permease – lacA = galactoside transacetylase • All 3 genes are transcribed together producing 1 mRNA, a polycistronic message that starts from a single promoter – Each cistron, or gene, has its own ribosome binding site – Each cistron can be translated by separate ribosomes that bind independently of each other
  • 153. 7-4 Control of the lac Operon • The lac operon is tightly controlled, using 2 types of control – Negative control, like the brake of a car, must remove the repressor from the operator - the “brake” is a protein called the lac repressor – Positive control, like the accelerator pedal of a car, an activator, additional positive factor responds to low glucose by stimulating transcription of the lac operon 7-5 Negative Control of the lac Operon • Negative control indicates that the operon is turned on unless something intervenes and stops it • The off-regulation is done by the lac repressor – Product of the lacI gene
  • 154. – Tetramer of 4 identical polypeptides – Binds the operator just right of promoter 7-6 lac Repressor • When the repressor binds to the operator, the operon is repressed – Operator and promoter sequence are contiguous – Repressor bound to operator prevents RNA polymerase from binding to the promoter and transcribing the operon • As long as no lactose is available, the lac operon is repressed 7-7 Negative Control of the lac Operon 7-8 Inducer of the lac Operon • The repressor is an allosteric protein – Binding of one molecule to the protein changes
  • 155. shape of a remote site on that protein – Altering its interaction with a second molecule • The inducer binds the repressor – Causing the repressor to change conformation that favors release from the operator • The inducer is allolactose, an alternative form of lactose 7-9 Inducer of the lac Operon • The inducer of the lac operon binds the repressor • The inducer is allolactose, an alternative form of lactose 7-10 Discovery of the Operon During the 1940s and 1950s, Jacob and Monod studied the metabolism of lactose by E. coli •Three enzyme activities / three genes were induced together by galactosides
  • 156. • Constitutive mutants need no induction, genes are active all the time • Created merodiploids or partial diploid bacteria carrying both wild-type (inducible) and constitutive alleles 7-11 Discovery of the Operon • Using merodiploids or partial diploid bacteria carrying both wild-type and constitutive alleles distinctions could be made by determining whether the mutation was dominant or recessive • Because the repressor gene produces a repressor protein that can diffuse throughout the nucleus, it can bind to both operators in a meriploid and is called a trans-acting gene because it can act on loci on both DNA molecules • Because an operator controls only the operon on the same DNA molecule it is called a cis-acting gene
  • 157. 7-12 Effects of Regulatory Mutations: Wild-type and Mutated Repressor 7-13 Effects of Regulatory Mutations: Wild-type and Mutated Operator with Defective Binding 7-14 Repressor-Operator Interactions • Using a filter-binding assay, the lac repressor binds to the lac operator • A genetically defined constitutive lac operator has lower than normal affinity for the lac repressor • Sites defined by two methods as the operator are in fact the same 7-15 The Mechanism of Repression
  • 158. • The repressor does not block access by RNA polymerase to the lac promoter • Polymerase and repressor can bind together to the lac promoter • Polymerase-promoter complex is in equilibrium with free polymerase and promoter 7-16 lac Repressor and Dissociation of RNA Polymerase from lac Promoter • Without competitor, dissociated polymerase returns to promoter • Heparin and repressor prevent reassociation of polymerase and promoter • Repressor prevents reassociation by binding to the operator adjacent to the promoter